ABSTRACT Title of Disertaion: Role of ubiquitination in Caenorhabditis elegans development and transcription regulation during spermatogenesis Madhura Kulkarni, Doctor of Philosophy, 2008 Disertation Directed by: Dr. Harold Smith Center for Advanced Research in Biotechnology Regulation of gene function can be achieved through a variety of mechanisms. In this disertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initialy identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an esential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive alele, (uba-1)it129 ts , was isolated in a clasical genetic scren for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microaray screning for sperm-enriched genes led to the cytological analysis of the deletion alele spe-44(ok1400), by reverse genetics approach. it129 ts encodes a conditional alele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necesary for al subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typicaly results in target protein degradation, which provides an esential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normaly targeted for proteasomal degradation instead persist in uba-1(it129 ts ) and impair critical celular proceses. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expresion during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion alele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arest of spermatocytes prior to spermatid diferentiation. spe-44 mRNA is expresed in a narow spatial and temporal window, just prior to spermatocyte diferentiation, consistent with its functional role during spermatogenesis. Future study wil be directed to find putative targets of spe-44 and the mechanisms that regulate gene expresion using microaray analysis and yeast-one hybrid screns. These studies wil help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans. ROLE OF UBIQUITINATION IN CAENORHABDITIS ELEGANS DEVELOPMENT AND TRANSCRIPTION REGULATION DURING SPERMATOGENESIS By MADHURA KULKARNI Disertation Submited to the Faculty of the Graduate School of the University of Maryland, College Park, in Partial Fulfilment of the Requirements for the Degre of Doctor of Philosophy 2008 Advisory Commite: Profesor Stephen Mount, Chair Profesor Harold Smith Profesor Eric Hag Profesor Caren Chang Profesor Jonathan Dinman Profesor Douglas Julin ? Copyright by MADHURA KULKARNI 2008 ii To My Parents and Gurus iii PREFACE Declaration of author?s intent to use own previously published text. The main text, tables, figures and figure legends in their entirety for: Chapter 2: E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout C. elegans development were used, and only modified to met the formating requirements of this disertation. Full citation is given below: Madhura Kulkarni and Harold Smith. E1 ubiquitin-activating enzyme plays multiple roles throughout C. elegans development. PLoS Genetics. 2008. Jul 18;4(7):e1000131 iv ACKNOWLEDGEMENTS I expres my profound gratitude to my advisor Dr. Harold Smith, whose guidance and constant encouragement have been a great source of inspiration in my profesional life. I consider myself fortunate to have had an opportunity to work with him. My sincere thanks to Dr. Stephen Mount, for being the chair of my disertation advisory commite and the constant support throughout. I also would like to thank my commite members Dr. Douglas Julin, Dr. Caren Chang. Dr. Jonathan Dinman and Dr. Eric Hag for their suggestions and critical evaluation of the work throughout the duration of my graduate study. I particularly acknowledge Dr. Todd Cooke, the ex-Graduate Director of the Department of Cel Biology and Molecular Genetics, for alowing me to work in Center for Advanced Research in Biotechnology. I?l also like to thank Dr. Zhongchi Liu, without whose support I couldn?t have been in this graduate program. I thank my lab members Dr. Daya Jirage, Dr. Antonio Del Castilo, Dr. Arun Subramanian, Sara Hapip, and Carina Nhe for their substantial contributions in terms of discussions, critique, development of the experiments, comments on my scientific writing, and encouragement during my graduate work. The worm club members at NIH, UMBC and JHU and the worm metings have been very helpful to keep a steady direction for my research. I need to thank especialy Dr. Kevin O?Connel, Dr. Andy Golden, and Dr. Chris Richie at NIH for their valuable time during my visits to NIH for the use of their facilities. v CARB is a unique place, which provided me a very comfortable environment during the last four years. I would like to thank everyone who made me fel at home, with the specific mention of the third floor group, Dr. Zvi Kelman, Dr. Jim Ames, Jae- Ho, Gun-Young, Jennifer, Jayanti, Alexandra and Larik. I appreciate the third floor metings for the insightful discusions and comments and regular potlucks. I expres my gratitude towards al my friends who have been part of my life in diferent eras during my (five year long) graduate journey. It?s impossible to mention how each one of them has been important during this time. Nozomi, Rajesh, Aswani, Supratik and Tushar deserve special mention. I would like to thank my roommates Sudeshna, Uma, Sankalita, Dena, Brian, Manish and Bob for being my home away from home. Al these people wil always be part of my enumerable memories from graduate school. I take this opportunity to say how imensely grateful I am to my parents Suneeta and Datatraya, my sister Mrunal and my fianc? Vikash, for their motivation, love and emotional support throughout. Madhura D Kulkarni vi TABLE OF CONTENTS Preface..........................................................................................................................iii Acknowledgements.......................................................................................................iv Table of contents..........................................................................................................vi List of Tables................................................................................................................ix List of Figures................................................................................................................x Chapter 1 Regulation of gene expresion in C. elegans development.......................1 1.1 Introduction............................................................................................................1 1.2 Transcriptional regulation of gene expresion.........................................................5 1.2.1 Transcription networks for epidermal and muscle specification in C. elegans...............................8 1.2.2 Transcription regulation in C. elegans sex determination...............................................................13 1.3 Ubiquitin conjugation as gene regulatory mechanism...........................................17 1.3.1 Enzymes involved in ubiquitin-conjugation....................................................................................21 1.3.2 Ubiquitin activating enzyme (E1), at the apex of the ubiquitin-conjugation pathway..................24 1.3.3 Ubiquitin-like (Ubl) modifiers and cros-talk betwen ubiquitin and Ubl conjugation systems.26 1.3.4 Ubiquitination in C. elegans development.......................................................................................27 1.3.5 Ubiquitination role in polarity establishment in early embryo.......................................................29 1.4 Interplay betwen transcriptional regulation and ubiquitin-mediated regulation....30 1.4.1 Interplay during the development of an early embryo of C. elegans.............................................31 1.4.2 Interplay during sex determination of C. elegans............................................................................31 Chapter 2 E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout C. elegans development...............................................................................................38 2.1 Abstract................................................................................................................38 2.2 Introduction..........................................................................................................39 vii 2.3 Materials and methods..........................................................................................42 2.3.1 Genetics..............................................................................................................................................42 2.3.2 Microscopy.........................................................................................................................................43 2.3.3 Sperm asays......................................................................................................................................43 2.3.4 Cloning and molecular analysis........................................................................................................44 2.4 Results..................................................................................................................46 2.4.1 Phenotypic characterization..............................................................................................................46 2.4.2 Male-specific phenotypes..................................................................................................................51 2.4.3 Sperm-specific defect of uba-1 mutation.........................................................................................57 2.4.4 Identification of it129 as uba-1.........................................................................................................61 2.4.5 In vivo defects in ubiquitination and embryogenesis.......................................................................69 2.5 Discussion............................................................................................................75 Chapter 3 spe-44, a putative transcription regulator of sperm gene expresion....82 3.1 Introduction..........................................................................................................82 3.1.1 Sperm development in C. elegans....................................................................................................83 3.1.2 Mutational analysis of spermatogenesis in C. elegans....................................................................89 3.1.3 Putative transcriptional regulators expresed during spermatogenesis..........................................92 3.2 Materials and Methods.........................................................................................94 3.2.1 Strains.................................................................................................................................................94 3.2.2 Single Worm PCR..............................................................................................................................95 3.2.3 Worm icroscopy..............................................................................................................................97 3.2.4 RNAi for C25G4.4.............................................................................................................................97 3.2.5 In situ hybridization...........................................................................................................................98 3.2.6 Western analysis..............................................................................................................................100 3.2.7 Microinjection rescue......................................................................................................................101 3.3 Results................................................................................................................102 3.3.1 C25G4.4 RNAi produces no phenotype.........................................................................................103 3.3.2 Deletion in C25G4.4 causes sperm-specific sterility.....................................................................105 3.3.3 Balancer and marker linked strains for spe-44...............................................................................106 3.3.4 Sperm-specific defect......................................................................................................................108 3.3.5 Sperm development is arested in meiosis.....................................................................................110 3.3.6 Spermatogenic fate is determined in spe-4(ok140) germline....................................................117 3.3.7 spe-44 is expresed in pachytene germline....................................................................................119 3.4 Discussion..........................................................................................................122 Chapter 4 Discusion..............................................................................................132 vii Chapter 5 Apendices............................................................................................140 5.1 Western analysis of uba-1(it129) worms to detect ubiquitin-activating enzyme expresion and ubiquitination of proteins.....................................................................140 5.1.1 Introduction......................................................................................................................................140 5.1.2 uba-1 cDNA cloning and in vitro protein induction......................................................................141 5.1.3 Western blot analysis with anti-E1 (ubiquitin-activating enzyme) antibody...............................144 5.1.4 estern analysis to detect ubiquitination levels in wild type and it129 ts mutant worms...........151 5.1.5 Western blot analysis with SP56 antibody.....................................................................................155 5.2 Genetic interaction betwen elt-1(zu180) and uba-1(it129 ts )...............................157 5.2.1 Introduction......................................................................................................................................157 5.2.2 elt-1 RNAi on early larval males leads to developmental defects in their tail organs.................158 5.2.3 elt-1 (zu180) homozygous strain behaves diferently at 15 0 C and 25 0 C......................................161 5.2.4 in situ hybridization to check elt-1 mRNA expresion patern in sperm producing germline...163 5.2.5 Complementation test betwen elt-1(zu180) and it129 ts ...............................................................167 5.2.6 elt-1 RNAi or wild type elt-1 genomic region microinjection in uba-1(it129 ts ) hermaphrodites leads to ?intersex? phenotype........................................................................................................................168 Bibliography..............................................................................................................172 ix LIST OF TABLES Table 2-1: Summary of uba-1(it129) phenotypes...........................................................47 Table 2-2: Maternal and paternal rescue of lethality......................................................50 Table 2-3: Synthetic interactions betwen double mutants.............................................72 Table 3-1: Putative sperm-gene regulators in C. elegans genome...................................93 Table 5-1: Proportion of genotypes in the F1 progeny from the strain carying elt- 1(zu180)..............................................................................................................162 x LIST OF FIGURES Figure 1-1: Specification of C lineage.............................................................................9 Figure 1-2: A Cartoon of the C lineage transcriptional network.....................................12 Figure 1-3: Schematic representation of cel-specific regulation of sexual fate by TRA-1. ..............................................................................................................................16 Figure 1-4: Schematic representation of ubiquitin-conjugation pathway........................22 Figure 1-5: Sex-specific regulation of xol-1...................................................................33 Figure 1-6: Ilustration of sex determination pathway components................................35 Figure 2-1: Defects in uba-1 hermaphrodites.................................................................49 Figure 2-2: Defects in uba-1 males................................................................................53 Figure 2-3: Sperm defects..............................................................................................60 Figure 2-4:Cloning and complementation......................................................................63 Figure 2-5: In situ hybridization of gonads....................................................................68 Figure 2-6: Western blot for ubiquitin...........................................................................70 Figure 2-7: OMA-1::GFP expresion.............................................................................74 Figure 3-1: Sperm development in wild type worm.......................................................84 Figure 3-2: Progresion of germline diferentiation in the gonad during spermatogenesis. ..............................................................................................................................86 Figure 3-3: Germline distribution and morphology of hermaphrodite and male gonad...88 Figure 3-4: Schematic representation of mutations at their respective arest points during spermatogeneis......................................................................................................91 xi Figure 3-5: Cartoon of the primers used to detect C25G4.4 deletion..............................96 Figure 3-6: Alignment of SAND domain from homologous proteins...........................104 Figure 3-7: ok1400 deletion is linked with sperm-specific sterility...............................107 Figure 3-8: Adult hermaphrodite spermatheca.............................................................109 Figure 3-9: Proximal region of L4 hermaphrodite gonad.............................................111 Figure 3-10: DIC micrograph of disected gonads.......................................................112 Figure 3-11: spe-44 terminal spermatocytes with 4 condensed nuclei..........................114 Figure 3-12: Early meiotic progresion of spe-44 germline..........................................115 Figure 3-13: Spermatocyte swep from the spe-44 young hermaphrodites...................116 Figure 3-14: Western analysis of spe-44 males with Anti-MSP...................................118 Figure 3-15: In situ hybridization on disected gonads with spe-44 anti-sense probe....120 Figure 3-16: Spatial patern of spe-44 expresion in the fem-3 adult germ line with respect to cel cycle stage................................................................................................121 Figure 3-17: CLUSTALW 2.0.5 multiple sequence alignment.....................................125 Figure 3-18: SPE mutations with terminal spermatocyte phenotype.............................129 Figure 5-1: Coomasie gel with crude protein extract from bacterial pelet expresing Ce- UBA-1.................................................................................................................143 Figure 5-2: Western analysis of total protein from wild type and uba-1(it129 ts ) worms with Anti-E1........................................................................................................148 Figure 5-3: Western analysis of total protein from isolated sperm for E1.....................150 Figure 5-4: Standardization of Western analysis with anti-ubiquitin antibody..............153 Figure 5-5: Western analysis with anti-ubiquitin antibody on protein extracts from adult worms and isolated sperm....................................................................................154 xii Figure 5-6: Western analysis with SP56 antibody on sperm protein extract.................156 Figure 5-7: Comparison of male tail structures betwen wild type and elt-1 RNAi treated wild type males....................................................................................................160 Figure 5-8: In situ hybridization to detect elt-1 expresion in the germlines.................166 Figure 5-9: Strategy used to generate heterozygous elt-1(zu180) over (it129 ts ) worms.167 Figure 5-10: Intersex phenotype in transgenic uba-1(it129 ts ) hermaphrodites carying elt- 1 transgene..........................................................................................................170 1 Chapter 1 Regulation of gene expresion in C. elegans development 1.1 Introduction Development of a multicelular eukaryotic organism occurs through growth and diferentiation from a single cel, the zygote. During maturation it diferentiates into many cel types, each with specificaly alocated function. The diversity of cel types and the co-ordination of function are determined by the diferential expresion of genes within each cel type. Thus, proper development depends on the precise temporal and spatial control for the diferential expresion of thousands of genes. Each cel expreses only a subset of the genes from the genome. For example, muscle cels expres about 7-10% (~1500) of the total genes (~19800) in C. elegans (Kim at al., 2001; Roy et al., 2002). Thus, at any given time, only a fraction of the genome is expresed in a cel. Because of the eforts of genome sequencing, we know the gene database for most of the eukaryotic model organisms. Yet, how the diferential expresion of these genes in individual cel types is regulated at a precise time and in response to environmental cues is stil unclear. Significant amounts of research efort have been directed in the past few decades towards understanding the proces of gene regulation. The expresion begins when a gene is transcribed into mRNA, and ends when a functional protein product is no longer needed. The studies done so far reveal that the gene function is regulated at every 2 possible step i.e. transcription, post-transcription (e.g. splicing), translation, post- translation (e.g. phosphorylation) and epigenetic (e.g. chromatin level). The first level of control for any gene to be transcribed is the chromatin structure, which determines the acesibility of the DNA at a particular locus for transcription initiation. Chromatin structure is determined by various post-translational modifications of histone proteins. For example, DNA methylation is one of these factors asociated with transcription represion reviewed in (Miranda and Jones, 2007). Transcription regulation is one of the most extensively studied modes of regulation for gene expresion. The regulation is achieved by one or more transcriptional activator or represor proteins, which bind to the promoter region of the gene in a sequence-specific manner. The characteristic patern of gene expresion for a particular cel type is determined by a specific set of transcription factors contained in that cel. The activity of the transcription factor itself is controlled by co-factors and signaling molecules. How transcription is regulated wil be discussed in more detail in the next section. Once a gene is transcribed into a pre-mRNA, it is procesed by RNA splicing to remove the non-coding intronic sequences. In higher eukaryotic organisms, along with the introns, alternative exons can be selected preferentialy leading to more than one splice variant of a transcript. About 10% of the genes in C. elegans undergo alternative splicing (Kim et al., 2007a). This proces of alternative splicing provides a post- transcriptional regulation for gene function. Diferent isoforms translated from the alternative splice variants can have tisue-specific or diferent sub-celular localization, or varied kinetic performances. 3 A mature mRNA is regulated via untranslated regions (UTR) in the 3? and 5? regions caled 3?UTR and 5?UTR, respectively. These UTRs play roles in the transport, localization, and stability of the mRNA. Significance of the spatial patern of mRNA localization during development is wel documented for Drosophila embryogenesis. Specific localization of mRNAs like Oskar, Vasa and Tudor is esential for the establishment of the primordial germ cels in the Drosophila embryo (reviewed in Wiliamson and Lehmann, 1996). AREs, AU-rich elements in the 3? UTR, play a role in stabilizing the mRNAs through recruitment of RNA-binding proteins. This stabilization can be crucial for proper development; for example, diferentiation of the nervous system in Drosophila is achieved by stabilizing the gene gcm (glial cels mising) (Soustele et al., 2008). The sequences in the UTRs are also employed to regulate the initiation of translation from the mRNA. As mentioned earlier, Oskar mRNA is not translated unles it reaches the desired localization. Before its proper localization, translation is inhibited by Bruno protein, which binds to the Bruno response element in the 3?UTR of Oskar mRNA (Snee et al., 2008). The polyadenylated [poly(A)] tail at the 3? end of mRNA is also known to control translation initiation of respective RNAs. Cytoplasmic polyadenylation element binding (CPEB) protein, which binds to Cytoplasmic Polyadenylation Element (CPE), can either repres translation by binding to inhibitors or can activate translation by extending the length of the poly(A) tail (Pique et al., 2008). CPEB has ben shown to be esential in various proceses like germ cel development (Setoyama et al., 2007), cel division (Luitjens et al., 2000) and synaptic plasticity (reviewed in Richter, 2007). Poly(A) tail length at the 3? end of mRNA also controls the 4 stability of the transcript. Poly(A) tail longer than 30 As stabilizes mRNA by blocking the asembly of exonucleases involved in RNA degradation (Ford et al., 1997). The recently recognized world of smal, non-coding RNAs reveal new dimensions for regulation of gene expresion via RNA silencing and translation represion as reviewed by Kim, 2005. After translation, the protein itself is subjected to regulation of its function via various post-translational modifications. There are nearly 200 diferent types of post- translational modifications of proteins known, which create one to two-fold diversity in the proteome compared to the genome of the organism (Walsh et al., 2005). Covalent modifications of the side chains in proteins by phosphorylation, acylation or glycosylation can regulate celular localization and functional aspects (e.g. kinetic properties) of the proteins. For example, dual phosphorylation of MAP kinases increases their catalytic eficiency by 10 5 -fold. Some of the post-translational modifications provide an ?on? or ?of? switch for the signal transduction cascade as in the clasic example of the RAS signaling pathway involved in various functions during celular development (reviewed by Campbel et al., 1998). RAS proteins belong to the superfamily of monomeric GTPases and are biologicaly active only when they have a farnesyl lipid modification. The lipid modification is required to anchor the protein in the membrane and to relay the switch to the downstream cascade of cytoplasmic kinases. To achieve precise spatio-temporal regulation of gene function, the duration for which a protein remains functional in the celular system needs to be monitored. Inactivation of the protein product once it is no longer needed, is critical for proper development. A specific type of post-translational modification is devoted to degradation 5 of the protein after its function is fulfiled. Covalent conjugation of ubiquitin, a 76 amino acid peptide, to the target protein marks it for degradation via the 26S proteasome. The proces of ubiquitination of the target protein itself is a highly regulated enzymatic cascade as discussed in detail in section 1.3. The regulation of gene function is further refined by the cross-talk betwen and within diferent modes of regulation. Understanding these events not only provides insights into fundamental mechanisms of gene function regulation, but also can lead to answers for curing various diseases caused by mis-regulation. The next sections wil discuss transcriptional regulation and ubiquitin conjugation of proteins with respect to C. elegans development. 1.2 Transcriptional regulation of gene expresion The importance of regulation of gene expresion in controlling the developmental programs of an organism is wel appreciated. The precise patern of gene expresion determines specific developmental decisions. During development, commitment to a specific fate followed by a determined diferentiation program is achieved through an interdependent network of transcription factors. Al genes require the basic transcriptional machinery known as general transcription factors (GTFs) and the spatio-temporal regulation of activation and/or represion of a gene is exerted through gene-specific factors. Eukaryotic GTF includes the RNA Polymerase I (Pol I), TBP (TATA-box binding protein) and TAFs (TBP asociated factors). The role of TBP is to bind the core promoter, and TAFs asist TBP in this proces (reviewed in Le and Young, 1998). 6 Factors that relay the gene-specific signal to GTFs are caled mediators. Generaly, a specific mediator functions in a specific developmental pathway to coordinate gene-specific factors with GTFs. Mediator proteins are functionaly and structuraly conserved in eukaryotes and many of them have been identified in the C. elegans genome (Bourbon et al., 2004). For example, med-15 functions in asociation with sbp-1, a member of the SREBP (sterol regulatory element binding protein) family of transcription activators and it is required for faty acid homeostasis in C. elegans (Yang et al., 2006). Thus, mediator proteins serve an important function of integrating the regulatory signal to the basic transcription machinery as they can interact with both GTFs and gene-specific transcription factors. The focus of this section wil be on gene-specific transcriptional regulation. Many mutations have been studied so far which afect early development of invertebrates and metazoans. The majority of these mutations are encoded in transcription factors, emphasizing their importance in establishing cel commitment and patern formation. C. elegans is a simple model system that has provided valuable insights into the field of transcription regulation. In C. elegans, 934 of the total ~ 20,000 genes encode putative transcription factors (TFs) (Rece-Hoyes et al., 2005). Recent progres in high- throughput technologies like microaray analysis and yeast-one hybrid screning is helping to understand the intricate network of these transcription factors and their target genes in the specification of tisue diferentiation (Dupuy et al., 2007; Vermeirsen et al., 2007). In C. elegans, transcription is silenced at the global level during oogenesis before oocytes begin maturation. Global silencing is achieved by regulating the phosphorylation 7 status of the carboxy-terminus domain (CTD) of the large subunit of RNA polymerase I (Seydoux and Braun, 2006; Walker et al., 2007). Oocyte maturation signals the release of transcriptional block, which aids rapid gene activation after fertilization in the zygote (Walker et. al., 2007). The transcriptional block ocurs at a step after initiation but before transcription elongation. Transcription resumes in the zygote after fertilization at the four-cel stage during embryogenesis (Baugh et al., 2003; Seydoux and Fire, 1994). This transcription restart occurs only in the cels of the somatic lineage, while transcription in the germline lineage is delayed until the 100-cel stage (Martinho et al., 2004; Melo et al., 1996; Seydoux and Dunn, 1997). Extended transcriptional silencing in the germline lineage cels is maintained by the maternaly supplied protein PIE-1 (Ghosh and Seydoux, 2008; Melo et al., 1996; Seydoux et al., 1996). PIE-1 encodes a putative RNA-binding protein with two CCH zinc-finger motifs and has been shown to repres transcription in mamalian cel culture experiments (Batchelder et al., 1999). PIE-1 inhibits transcription by competing with P- TEFb, a kinase subunit which phosporylates CTD, thus blocking transcriptional elongation (Zhang et al., 2003). pie-1 null mutation leads to embryonic lethality due to precocious release of transcription suppresion in the germline cels, which as a result adopt the somatic fate (Melo et. al., 1996). When the embryo reaches the 100-cel stage, PIE-1 levels drop in germline cels (Melo et. al., 1996) due to the action of emb-4 via an unknown mechanism (Chechi and Kely, 2006). Thus, the commitment of cel lineage for somatic or germline fate is determined by temporal regulation of the global transcription restart in the embryo. 8 1.2.1 Transcription networks for epidermal and muscle specification in C. elegans As mentioned earlier, high-throughput techniques like microaray and yeast-one hybrid make it easier to study gene expresion at the global level and to decipher the link betwen specific transcription factors and the set of target genes. RNAi knockdown of genes in C. elegans provides an advantageous system to validate functional significance of these targets and transcription cascades in a context of fate determination or patern formation. A recently published study that revealed a multi-step cascade of gene activation and the interplay betwen the networks of transcription factors in distinguishing the cel fate of C blastomere descendents in C. elegans development (Yanai et al., 2008), is a very good example of these high-throughput regulation analyses. C. elegans embryogenesis and subsequent development is a characteristic example of invariant cel lineage with a fixed patern of cel divisions and a fixed developmental program defined for every daughter cel (Sulston et al., 1983). Through a series of asymmetric cel divisions, a founder cel population with each cel specified for a distinct lineage is generated by the 8-cel stage in the embryo. As shown in Figure 1-1, the AB lineage produces hypodermis, neurons, anterior pharynx and other cel types; MS produces the somatic gonad, muscle, the majority of the pharynx, neurons and gland cels; E produces al intestine; C produces muscle, hypodermis and neurons; D produces muscle; and the P4 cel is the germ-line precursor (G?nczy and Rose, 2005). The C blastomere, which further diferentiates into muscle, hypodermis and neuronal fates, is specified by the expresion of pal-1, a caudal homeoprotein (Hunter and Kenyon, 1996). In the absence of maternal PAL-1 activity, the C blastomere does not 9 Figure 1-1: Specification of C lineage. The C blastomere is born at the eight-cel embryonic stage (A-D) and divides asymmetricaly to produce muscle cels and epidermal cels (E). (Figure modified from G?nczy and Rose, 2005). 10 develop into these fates, while ectopic pal-1 translation causes other founder cels to produce muscle, epidermal and neuronal cels (Draper et al., 1996; Hunter and Kenyon, 1996). Initial microaray analysis for pal-1 targets revealed 308 candidate targets (Baugh et al., 2005). Functional annotations of these targets indicated 13 putative transcription factors representing many families, including homeodomain, zinc-finger, GATA, MADS domain, bHLH and T-box proteins. So many diverse transcription factors amongst the targets of PAL-1 suggested that pal-1 controls a transcriptional regulatory network (Baugh et al., 2005). Further, RNAi followed by microaray analysis and reporter gene expresion of pal-1 targets and putative transcription factors indicated two sub-networks of transcription regulators aranged in topological order (Yanai et. al., 2008). The interesting finding was that these two sub-networks compete with each other to specify either muscle or epidermal cel fate. Epidermal cel fate is induced by elt-1, a GATA transcription factor necesary and sufficient for determining epidermal fate (Gileard et al., 1999). Its expresion is activated by pal-1 in the epidermal lineage of C blastomere (Baugh et al, 2005). elt-1 in turn activates expresion of downstream transcription factors elt-3, lin-26 and nhr-25 (Baugh et al., 2005; Gileard et al., 1999; Labouese et al., 1996; Page et al., 1997). elt-1 itself is temporaly regulated, as its expresion peaks during the earlier stages of epidermal fate determination and is reduced later (Baugh et al., 2003). RNAi of elt-1 reduced target gene expresion, consistent with the genetic studies, but RNAi of any of these thre target genes elevated the expresion of elt-1 and the rest of the targets (Yanai et. al., 2008). Thus, the target transcription factors impart a negative fedback on their common activator, generating a transcriptional network for epidermal fate determination. This 11 topology of expresion suggests that epidermal specification occurs via a two-step proces, with a negative fedback loop. Unlike epidermal fate, muscle fate is specified by thre transcription factors; hlh- 1, hnd-1 and unc-120 (Baugh et al., 2005; Fukushige et al., 2006). Out of 81 total body wal muscles, 32 are born from the C lineage, and the rest are born from the AB, MS and D lineages (Sulston et al., 1983). hlh-1 is shown to be esential for muscle fate specification irespective of the lineage (Krause et al., 1994). Its activation is regulated by diferent factors in distinct cel lineages and pal-1 regulates its activation only in C and D cel lineages. The thre transcription factors mentioned above, hlh-1, hnd-1 and unc-120, act redundantly in muscle specification based on genetic studies. They sem to activate each other?s expresion as shown by RNAi (Yanai et. al. 2008). RNAi of hlh-1 reduces the expresion of hnd-1 and unc-120. RNAi of hnd-1 or unc-120 reduces the expresion of each other, but both increase the mRNA levels of hlh-1, indicating negative fedback loop similar to the one observed during epidermal fate determination. As summarized in Figure 1-2 these two networks of transcription regulators are initiated from the same cel lineage, yet specify a distinct cel fate. This distinction is achieved by antagonistic action of epidermal and muscle transcription networks on each other (Yanai et al., 2008). RNAi of any of the thre muscle TFs resulted in increased expresion of al four epidermal TFs, suggesting that the muscle network represes the epidermal network of transcription factors. Yeast-one hybrid analysis with the elt-1 promoter showed that 10 out of 13 transcription factors bound the promoter, indicating that the muscle network of transcription factors directly 12 Figure 1-2: A Cartoon of the C lineage transcriptional network. PAL-1 initiates C blastomere development, inducing first elt-1 such that ELT-1 is present in al cels at the 4-cel (4C) stage embryo. At 8-cel (8C) stage, ELT-1 induces the second stage epidermal TFs and represes the muscle TF network. In the posterior daughter cel, ELT-1 expresion is not maintained and al thre muscle TFs are expresed, which suppres elt-1 expresion (Modified from Yanai et al., 2008). 13 regulates elt-1 expresion. Similar and reciprocal increase in the expresion levels of muscle transcription factors was observed in elt-1 RNAi. During development, the C blastomere expreses muscle transcription factors and elt-1 expresion in the epidermal daughter cel is esential to suppres the muscle fate (Yanai et. al., 2008). 1.2.2 Transcription regulation in C. elegans sex determination C. elegans exists as two naturaly occurring sexes, hermaphrodite (with two copies of the X chromosome) and male (with one copy of the X chromosome). The hermaphrodite is somaticaly female but produces male gametes (sperm) for a short period of time. Both sexes exhibit numerous sex-specific diferences in the body plan. About 40% of male and 30% of hermaphrodite cels are sexualy specialized and lead to extensive sexual dimorphism in C. elegans (Sulston and Horvitz, 1977). These sex- specific diferences are orchestrated by diferential expresion of genes throughout development, as identified by the microaray analysis of global expresion levels in two sexes (Jiang et al., 2001). The master regulator, TRA-1, defines the diferential expresion in a sex-specific manner. It encodes a GLI family transcription factor with zinc fingers and its mRNA is expresed at similar levels in both the sexes of C. elegans (Zarkower and Hodgkin, 1992). Its function is required to initiate hermaphrodite fate and to suppres male fate in a tisue-specific manner. Diferential acumulation of TRA-1 protein leads to distinct sexual fates as shown by Schvarzstein and Spence (2006). This diferential acumulation of TRA-1 is achieved via sex-specific proteolysis regulated by intricate control of the sex-determination pathway, which is elaborated in the next section on ubiquitination 14 (Figure 1-6). In this section, I?l discuss its function as a global transcriptional regulator in directing sexualy dimorphic development in a tisue-specific manner (Summarized in Figure 1-3). The hermaphrodite-specific neurons (HSNs) are required for proper egg-laying behavior of hermaphrodites. They are born embryonicaly in both hermaphrodites and males but are retained only in hermaphrodites during the course of diferentiation. The HSNs are not needed in males and undergo programed cel death during embryonic development (Sulston and Horvitz 1977, Sulston 1983). The cel death is induced by pre- apoptotic gene egl-1, which encodes a protein with a BH3 domain known as cel death activator (Conradt and Horvitz, 1998). egl-1 is directly represed in hermaphrodites by TRA-1 (Conradt and Horvitz, 1999). As TRA-1 activity is reduced in males, egl-1 represion is released, inducing male-specific cel death of HSNs. Another clas of neurons that show sexualy-dimorphic programed cel death in C. elegans is the cephalic male neuron (CEM). The CEM neurons die during hermaphrodite embryogenesis but survive in males (Sulston et al., 1983). These neurons are esential for males to respond to the hermaphrodite pheromone (Chasnov et al., 2007). Survival of CEMs in males is regulated by ceh-30, an anti-apoptotic Bar- homeodomain transcription factor (Schwartz and Horvitz, 2007). Sex-specific expresion of ceh-30 is directly regulated by TRA-1 as it represes ceh-30 in hermaphrodites thereby alowing CEM death (Schwartz and Horvitz, 2007). A homolog of DM domain transcription factors in C. elegans, MAB-3, promotes development of male-specific organs and male mating behavior (Shen and Hodgkin, 1988; Yi et al., 2000). For example, in development of V rays, a sensory organ required 15 for male mating, mab-3 promotes activation of bHLH transcription factor lin-32 by preventing expresion of the Hes family transcription factor ref-1 specificaly in males (Ross et al., 2005). mab-3 is also required to suppres the vitelogenin gene vit-2 in male intestine. Thus, mab-3 primarily appears to function at the apex of the transcriptional regulatory cascade as a represor during male specific development. mab-3 is a target of TRA-1 as TRA-1 inhibits mab-3 expresion in hermaphrodites thus suppresing male- specific development (Yi et al., 2000). Along with the somatic sexual fate, TRA-1 is also required for germline sexual fate determination. The male germline continues spermatogenesis throughout its life while the hermaphrodite germline temporarily produces male gametes during L4 larval stage. In somatic tisue, TRA-1 acts as female fate-inducing factor by suppresing male- specific gene expresion. Genetic studies indicate that TRA-1 activity is esential in the germline for both oogenic and spermatogenic fate determination. So far, the only known direct target of TRA-1 in specifying the germline fate is fog-3 (Chen and Elis, 2000). fog-3 encodes a Tob homolog, and along with fog-1 promotes spermatogenesis in both sexes. Both have ben proposed to act as terminal regulators for sperm fate decision (Barton and Kimble, 1990; Elis and Kimble, 1995). TRA-1 promotes oogenesis in adult hermaphrodites by transcriptional represion of fog-1 and fog-3 (Chen and Elis, 2000; Jin et al., 2001; Lamont and Kimble, 2007). But TRA-1 also sustains continued spermatogenesis in males and in L4 stage hermaphrodites (Hodgkin, 1986; Hodgkin and Brenner, 1977; Kimble, 1988), most likely by positively regulating fog-3 under special 16 Figure 1-3: Schematic representation of cel-specific regulation of sexual fate by TRA-1. TRA-1, a GLI family transcription factor, regulates sexualy dimorphic development in a tisue-specific manner mainly by represing male-specific target genes in the hermaphrodite soma. 17 circumstances (Chen & Elis, 2000). No downstream targets of fog-3 and fog-1 are known as yet, and how these two genes bring about sperm diferentiation is stil an open question. In chapter II, I present the study of one of the putative transcription regulators that acts downstream of sperm-fate determination and is esential for sperm diferentiation. Even though transcriptional regulatory activity of TRA-1 is wel established, very few direct downstream targets of TRA-1 have ben identified. Sex-specific microaray experiments done by Jiang et al., (2001) identified 1,651 genes enriched in males and 520 genes enriched in hermaphrodites out of the total of 18,967 genes. These sex-regulated genes include 37 that encode putative transcription factors. Twenty-thre of these mRNAs that encode sex-regulated transcription factors are enriched in hermaphrodites, and fourten are enriched in males. Thre of these 37 putative transcription factors are shown to be direct targets of TRA-1 (Jiang et. al., 2001). This set of 37 putative transcription factors could be direct or indirect targets of TRA-1 and could constitute the regulatory network that controls sexualy dimorphic gene expresion paterns, which needs to be studied further. 1.3 Ubiquitin conjugation as gene regulatory mechanism Ubiquitin mediated degradation of TRA-1 has been identified recently as a critical mechanism for controlling its activity (se below). Ubiquitin, a 76 amino acid peptide, is conjugated to various proteins via a cascade of enzymatic reactions. Post-translational modification of proteins by ubiquitin conjugation regulates gene function mainly by controlling protein turnover mediated by a multisubunit 26S proteasomal complex 18 (Hershko and Ciechanover, 1998). More recently, ubiquitination has also ben shown to regulate protein activity and their spatial sorting. Ubiquitin conjugation plays an esential role in cel fate determination and organismal development. Aberant ubiquitin conjugation has been asociated with various cel cycle defects, developmental diseases and neuropathologies (Ardley and Robinson, 2004; Nakayama and Nakayama, 2006). Ubiquitin is translated as thre diferent precursors: a polymeric head-to-tail concatemer (polyubiquitin) and two N-terminal fusion proteins with ribosomal polypeptides UbL40 and UbS27 (Nenoi et al., 2000). Although more variations of ubiquitin encoding exist (Catic and Ploegh, 2005), these thre are the most conserved genes amongst phyla. Also, the number of polyubiquitin-encoding loci and the number of coding repeats found in the gene varies from species to species (Ozkaynak et al., 1984; Wiborg et al., 1985). The concentration of fre ubiquitin moieties in the cel is of critical importance and is monitored by regulating transcription from the polyubiquitin gene and release of ubiquitin monomers after degradation of the polyubiquitin tag from the conjugated proteins. This ubiquitin homeostasis is esential for cel survival and proper function of the proteasome, as failure to do so can lead to defects from meiotic arest (Okazaki et al., 2000) to various diseases (Hanna et al., 2007). The polyubiquitin gene produces the precursor polypeptide polyubiquitin. This unprocesed polyubiquitin chain has an additional amino acid residue at its C-terminus in the last ubiquitin moiety, which prevents its activation and hence conjugation to the target protein (Ozkaynak, et al., 1984). The polyubiquitin precursor is then procesed by de- ubiquitinating enzyme to release monomeric ubiquitins (Johnston et al., 1999). 19 Regulation at both the transcriptional step and the procesing step can control the availability of fre ubiquitin monomers, thus maintaining homeostasis. The monomeric ubiquitin has a Gly-Gly sequence at the C terminus, which then is activated by ubiquitin-activating enzyme. It is an ATP-dependent proces caried out by the ubiquitin-activating enzyme (E1) in two steps: 1) adenylation of the C terminus of ubiquitin polypeptide through the hydrolysis of ATP, and 2) the transfer of this ubiquitin to a conserved cysteine residue within E1. As a result, ubiquitin gets covalently atached to the cysteine via a thioester linkage, generating ubiquityl-S-E1 (Has and Rose, 1982). The activated ubiquitin is then transfered to ubiquitin-conjugating enzyme, E2, via energy-neutral trans-esterification (Ciechanover et al., 1982; Pickart and Rose, 1985). The E2 enzyme then transfers the ubiquitin moiety to the substrate protein either directly or with an additional step of an E3 ubiquitin ligase, a proces refered to as ubiquitination of the target protein. Ubiquitination of the substrate leads to branched-protein conjugates in which the C-terminal glycine residue of ubiquitin is linked by an isopeptide bond to a specific internal lysine residue (aceptor lysine) (Ciechanover and Schwartz, 1989; Hershko, 1991; Reis et al., 1989). Multiple rounds of this cascade of enzymatic reactions lead to a polyubiquitin tag on the target protein. The majority of substrates conjugated with ubiquitin are subjected to proteolysis via the 26S proteasome (reviewed in Hershko, 1991; Jentsch, 1992; Rechsteiner, 1987). Thus, post-translational modification with ubiquitin mainly regulates half-life of the substrate protein. Ubiquitin has seven lysine residues - K 6 , K 11 , K 27 , K 29 , K 33 , K 48 and K 63 . Polyubiquitin chains built up on distinct lysine linkages difer in their structural and functional information. Regioselectivity for specific lysine residue of ubiquitin as an 20 aceptor site for the first covalent atachment to a substrate depends on the ubiquitin- conjugating enzyme, E3, and the nature of the substrate. K 48 and K 63 are the best- characterized residues involved in polyubiquitylation (Haglund and Dikic, 2005). A polyubiquitin chain of at least four ubiquitin molecules at K 48 is sufficient to target a conjugated substrate for proteasomal degradation (Thrower et al., 2000). In contrast, K 63 linkage is involved in non-proteolytic functions like regulation of celular proceses such as DNA repair, signal transduction, intracelular traficking, and ribosomal biogenesis (Chan and Hil, 2001). Recently, novel non-canonical functions of K 48 and K 63 linkages have been discovered (reviewed in Li and Ye, 2008). Met4 activates expresion of genes in the methionine biosynthetic pathway in S. cerevisiae. Its transcriptional activity is regulated by polyubiquitination via K 48 but in non-proteolytic fashion (Kaiser et al., 2000). Similarly, canonicaly K 63 linkage is involved in non-proteolytic functions, but it has also been implicated to target substrates for proteasomal degradation (Li and Ye, 2008). Unlike K 48 and K 63 linkages, which have been studied extensively, very litle is known about the biological significance of other ubiquitin linkages. For example, Deltex, an E3 ligase involved in the Notch signaling pathway, itself gets polyubiquitinated by another E3 ligase, AIP4 via K 29 likage and is then targeted for lysosomal degradation (Chastagner et al., 2006). AMPK-related kinases regulate cel polarity and proliferation. NUAK1 and MARK4 are members of this family and are regulated via K 29 /K 33 -linked polyubiquitin chains (Al-Hakim et al., 2008). Recent studies also report mixed ubiquitin 21 chains that contain more than one type of ubiquitin linkage within a single polymer (Ben- Sadon et al., 2006; Kim et al., 2007b). Apart from the polyubiquitin tag, the E1-E2-E3 enzymatic cascade also regulates monoubiquitination and multiple monoubiquitinations of the target protein. The addition of a single ubiquitin to a substrate is refered as monoubiquitination (Reveiwed in Hicke, 2001). It plays a regulatory role in the endocytosis of plasma membrane proteins, DNA repair, histone modification and transcriptional regulation (Hicke, 2001). When multiple lysine residues in the substrate are conjugated to monoubiquitin moieties it is refered as multiple monoubiquitination (Haglund et al., 2003). 1.3.1 Enzymes involved in ubiquitin-conjugation The enzymes involved in the ubiquitin-conjugation are described in detail from the bottom of the cascade. The substrate selectivity and type of ubiquitin linkage is determined by an E3 ubiquitin ligase. The E3s are a large, diverse group of proteins defined by diferent characteristic motifs. The majority of them can be grouped in two categories, the RING (realy interesting new gene) clas and the HECT (homologous to E6-asociated protein C-terminus) clas (reviewed in Jentsch et al. 1992). RING E3s function as mediators betwen E2 and the substrate. They interact with both simultaneously, bringing the substrate lysine in close proximity to the reactive ubiquitin- E2 thioester bond and facilitating the transfer of the active ubiquitin. Thus, RING E3s work as adaptors and do not posses catalytic activity. These RING E3s can work as stand-alone adaptors like Mdm2 or in huge complexes like APC, the anaphase-promoting complex. HECT E3s have a catalytic function. The ubiquitin is first transfered from the 22 Figure 1-4: Schematic representation of ubiquitin-conjugation pathway. Ubiquitin-activating enzyme (E1) activates ubiquitin in ATP-dependent reaction. E1 interacts with multiple ubiquitin-conjugation enzymes (E2s) to transfer the activated ubiquitin-thiolester. E2 can transfer the ubiquitin to multiple ubiquitin ligases (E3s) which then ubiquitinate specific target proteins. 23 E2 to an active-site cysteine in the conserved HECT domain of the E3. The thioester- linked ubiquitin is then transfered to substrate from E3 (Schefner et al., 1995). HECTs play important roles in disease-related pathways like TGF-? signaling and p53 regulation (reviewed in Ke and Huibregtse, 2007). Ubiquitin conjugation enzymes (UBCs) known as E2s, function at the intermediate step of the ubiquitination cascade, transfering activated ubiquitin from E1 to either E3 or directly to a substrate with the help of an E3. Recent studies suggest that E2 interacts with E1 and E3 in mutualy exclusive fashion, such that E1 must be disociated after ubiquityl transfer before E2 can bind a specific E3 (Eletr et al., 2005; Huang et al., 2005). Al known E2 enzymes have a conserved 16Kd UBC domain. This domain contains a centraly located cysteine residue esential for ubiquitin-thiolester formation (Sung et al., 1991). The cascade of ubiquitin conjugation through a series of enzymes is driven by the diferential afinities of the E1-E2 and E2-E3 enzymes towards each other in ubiquitin- linked and ubiquitin-unlinked state. Ubiquitin-thioestered E1 has higher afinity for fre E2. Once the E2 is coupled to ubiquitin, E1 looses its afinity for ubiquitin linked E2 (Hershko et al., 1983; Pickart and Rose, 1985). Similar afinity variations have been demonstrated for E2-E3 interactions (Kawakami et al., 2001; Siepmann et al., 2003). Ubiquitin-dependent regulation of protein activity and degradation is an esential function of development and survival of an eukaryotic system. Vast numbers of substrate proteins are regulated by ubiquitin conjugation, and they are involved in diverse proceses like DNA repair, cel cycle progresion, transcriptional regulation, receptor- mediated signaling pathways, and stres response (Petroski and Deshaies, 2005; 24 Weisman, 2001). The diversity is achieved by ubiquitin ligases with their remarkably specialized substrate recognition, and there are over 100 diferent E3s known in various eukaryotic systems (Hicke et al., 2005). E2s are encoded by relatively lower number of proteins; for example, the S. cerevisiae genome encodes 13 UBCs while the human genome contains 30 putative UBCs (Schefner et al., 1998). 1.3.2 Ubiquitin activating enzyme (E1), at the apex of the ubiquitin- conjugation pathway In contrast to E3s and E2s, there is typicaly only one E1 protein, which serves the ubiquitin-activating function. Exceptions include an additional spermatogeneis-specific E1 in rodents, marsupials and human testis (Kay et al., 1991; Mitchel et al., 1992; Zhu et al., 2004) and multiple E1s encoded in plant genomes (Hatfield et al., 1997; Hatfield and Vierstra, 1992). The E1 enzyme contains signature motifs, two UBACT domains for ubiquitin activation and one or two Thif domains. The cysteine residue upstream of the UBACT domain is the most conserved residue as it is esential for covalent atachment of ubiquitin (Hatfield et al., 1992). Multiple forms of E1 protein have been detected in animals and plants (Cook and Chock, 1992; Hatfield et al., 1997). These diferent isoforms have been shown to vary in their post-translational modifications and sub- celular localization (Trausch et al., 1993). For example, the human E1 gene encodes two isoforms, E1a of 117KDa size and E1b of 110KDa size (Cook and Chock, 1992; Cook and Chock, 1995; Handley-Gearhart et al., 1994). Only the E1a isoform is shown to be dynamicaly phosophorylated in human cel lines (Stephen et al., 1996), and phosophorylated E1a isoform predominantly localizes in the nucleus during G2 phase of 25 cel cycle (Stephen et al., 1997). Quantitative distribution studies in the nucleus and the cytosol of HeLa cels suggested that the concentration of functional E1 (presumably the phosphorylated form) is the rate-limiting step for ubiquitin conjugation in the nucleus (Stephen et al., 1997). Although more than one isoform of E1 exists, there is esentialy one functional E1 in the system. Thus, the complexity of ubiquitin conjugation realized at the level of E3 function narows down to the single enzyme, E1, esential for ubiquitin activation. As the first enzyme in the pathway, E1 has the potential to regulate the rate of ubiquitin conjugation (Hatfield et al., 1990; Stephen et al., 1996). Blocking the E1 function can collapse the entire downstream ubiquitin conjugation cascade, as has been demonstrated in diferent species by various temperature-sensitive aleles of E1. Genetic studies in the yeast S. cerevisiae have revealed that inactivation of the yeast E1 gene, Uba1, blocks most of the ubiquitin conjugation (Ghaboosi and Deshaies, 2007; McGrath et al., 1991; Swanson and Hochstraser, 2000). Chapter I of this disertation also reports that a hypomorphic alele of uba-1 in C. elegans dramaticaly reduces the amount of ubiquitin conjugates globaly in total protein extracts (Kulkarni et al., 2008). Since functional aberations in E1 activity afect al possible ubiquitin conjugation reactions, conditional mutations in E1 have turned out to be extremely useful in uncovering and understanding ubiquitin-dependent functions. Mamalian cel lines containing temperature-sensitive aleles of E1 have revealed its functional role in cel cycle progresion and ubiquitin-mediated proteolysis (Ciechanover et al., 1984; Ciechanover et al., 1985; Finley et al., 1984; Kulka et al., 1988; Salvat et al., 2000). A conditional E1 mutant in S. cerevisiae has helped elucidate the mechanism of 26 polyubiquitin chain recognition by proteasomal components (Ghaboosi and Deshaies, 2007). Studies with weak and strong E1 aleles in Drosophila show opposing efects on cel survival, revealing complexities of the ubiquitin conjugation pathway (Le et al., 2008). In that study, partial loss of ubiquitin conjugation caused by weak Uba1 aleles inhibited cel death, while strong Uba1 aleles showed high apoptotic activity. At the same time, the strong alele induced neighboring cel proliferation in non-autonomous manner due to failure to downregulate Notch signaling (Le et al., 2008). 1.3.3 Ubiquitin-like (Ubl) modifiers and cros-talk between ubiquitin and Ubl conjugation systems Along with ubiquitin, there is family of ubiquitin-like modifiers (Ubls), which are conjugated to target proteins in a similar E1-E2-E3 conjugation cascade. A series of Ubls (e.g. SUMO, NED8, UCRP, FAT10, HUB, Fau, APG12, URM1, ISG15, Atg8) are emerging from recent studies. Some of them share sequence homology with ubiquitin but the majority of them have similar 3D topology, caled the ?-grasp fold (reviewed by Hochstraser, 2000; Jentsch and Pyrowolakis, 2000; Kerscher et al., 2006). They function as critical regulators of distinct celular proceses like transcription, DNA repair, signal transduction, autophagy, and cel-cycle, but via non-proteolytic mechanisms except for FAT10 (Reviewed in Kerscher et al., 2006; Schwartz and Hochstraser, 2003). Each of these Ubls has a dedicated E1 and E2 for its activation and conjugation as reviewed in Kerscher et. al., (2006). So far it had been thought that conjugation of distinct Ubls occurs through paralel and non-identical enzymatic cascades. But in last few years, emerging cross-talk betwen the components of distinct 27 Ubl?s machinery is chalenging this concept. For example, a common E2, UbcH8, is involved in conjugation of two distinct polypeptides; ISG15 and ubiquitin. The E1 enzyme transfers ubiquitin onto UbcH8, whereas the Ube1L/E1ISG15 transfers ISG15 onto UbcH8 (Zhao et al., 2004). Another example of crosstalk is Atg8 and Atg12, which are involved in autophagosome formation. Both share a single E1, Atg7, but each Ubl uses a distinct E2 (Ichimura et al., 2000). SUMO-1, 2, and 3 also share a common E1, a heterodimer of AOS1-UBA2 (Johnson, 2004). UBA6, an E1, ubiquitin-activating enzyme-like protein, is shown to activate both ubiquitin and FAT10 (Chiu et al., 2007). Even more interestingly, it transfers conjugated ubiquitin only to a specific subset of E2s, to Ubc5 and Ubc13 but not to Ubc3 and E2-25K (Chiu et al., 2007). Thus, ubiquitin- activating enzyme is not the sole activator of ubiquitin and this finding increases the complexity of ubiquitin-mediated regulation. 1.3.4 Ubiquitination in C. elegans development As in other eukaryotic organisms, ubiquitin-mediated protein regulation plays important roles in multiple aspects of C. elegans development. Detailed lists of the known and putative components of the ubiquitin conjugation pathway are wel summarized by Kipreos (2005) and by Jones et al., (2002). The C. elegans genome has two ubiquitin loci; ubq-1, a polyubiquitin locus (Graham et al., 1989), and ubq-2, which encodes ubiquitin fused to the L40 ribosomal large subunit protein (Jones and Candido, 1993). The ubq-1 locus encodes 11 tandem repeats of ubiquitin as an 838 amino acid polypeptide. The genome encodes over 600 putative E3s, 22 E2s, thre E2 variants without the critical catalytic cysteine, one E1, and four E1-like proteins. More than the 28 list of the factors, functional aspects of ubiquitin-conjugation pathway in C. elegans development wil be discussed further. Knock down by RNAi of uba-1, which encodes the sole E1 ubiquitin-activating enzyme, leads to severe phenotypes. The treated worms die earlier than wild type without producing any fertilized embryos (Jones et al., 2001). This emphasizes that ubiquitin conjugation is esential for progeny formation and functions through adult maintenance. The conditional alele of uba-1, reported in this disertation, also points towards the same conclusion based on the various diverse roles uncovered in the mutant worms (chapter I and Kulkarni et al., 2008). Only four of the 22 UBCs, let-70 (ubc-2), ubc-9, ubc-12 and ubc-14, play esential roles during early embryonic development as shown by RNAi experiments and by elevated expresion in microaray experiments (Jones et al., 2001). Depletion of ubc- 20 by RNAi brings developmental arest at the L3 larval stage, indicating its functional necesity during larval development (Jones et al., 2001). RNAi depletion of the rest of the UBCs individualy does not impair any aspect of C. elegans development (Jones et al., 2001). This observation is most likely due to the fact that E3s can interact with more than one E2; thus, E2s could be serving redundant functions in specific developmental pathways. This notion is also implicated in yeast-two hybrid studies for interactions within the ubiquitin conjugation system of C. elegans (Gudgen et al., 2004). Involvement of ubiquitin-mediated regulation in individual developmental aspects of C. elegans is disected through studies of the terminal E3 ligases. Although not al predicted E3s have been studied at the genetic or biochemical level, a number of them are known to regulate various developmental roles, as reviewed in WormBook (Kipreos, 29 2005). Recent studies have uncovered E3s playing functional roles in aging (Li W et al., 2007), synaptic signaling and plasticity (Schaefer and Rongo, 2006; Teng and Tang, 2005), endoplasmic reticulum-asociated degradation (Sasagawa et al., 2007), and sex determination (Jager et al., 2004; Starostina et al., 2007) in C. elegans. 1.3.5 Ubiquitination role in polarity establishment in early embryo The role of ubiquitin-mediated degradation is evident from the first cel division of the zygote. The posterior cel generated after the first asymmetric division of the zygote is dedicated for germline, whereas the anterior cel specifies somatic fate. The asymmetry is determined and established by multiple protein and RNA factors. CCH finger-encoding proteins, like PIE-1, POS-1 and MEX-1 bind RNA, and are known to segregate preferentialy to the germline cel during the first cel division (Guedes and Pries, 1997; Tenenhaus et al., 1998). The restriction of these CCH finger proteins to the germline lineage requires their degradation in somatic lineage cels. Degradation specificaly in the somatic lineage is mediated by a zinc finger-interacting protein, ZIF-1 (DeRenzo et al., 2003). ZIF-1 acts as a substrate recruitment factor and regulates PIE-1, POS-1 and MEX-1 degradation via CUL-2-containing E3 ligase (DeRenzo et al., 2003). The anaphase-promoting complex (APC), a multi-subunit E3 ligase, is involved in germline proliferation, cel cycle progresion in early embryo, and formation of the hermaphrodite vulva and male tail of C. elegans (Shakes et al., 2003). Its role in the metaphase-to-anaphase transition of meiosis I in C. elegans is very wel characterized. The APC has been shown to poly-ubiquitinate securin, the inhibitory partner of separase (Cohen-Fix et al., 1996; Funabiki et al., 1996). Once securin is degraded, separase is 30 relieved from the inhibition. Active separase then proteolyticaly cleaves cohesin, a protein that holds sister chromatids together, so that the sister chromatids can be pulled to opposing spindle poles (Buonomo et al., 2000; Nasmyth et al., 2000). Thus, the metaphase-to-anaphase transition is mediated by APC action. When any one of the subunits of APC is depleted either by RNAi or a genetic mutation, the afected embryos show a cel cycle block in metaphase of meiosis I or delayed progresion through meiosis (Davis et al., 2002; Furuta et al., 2000; Golden et al., 2000; Kitagawa et al., 2002; Shakes et al., 2003). Sex determination of C. elegans is also regulated by ubiquitination as wil be discussed in detail in the following section. 1.4 Interplay betwen transcriptional regulation and ubiquitin- mediated regulation Gene function is regulated at multiple levels as discussed in the introduction. These modes of regulation do not function as stand-alone mechanisms, but they in turn regulate each other?s function to create an intricate network for controling gene expresion. For example, monoubiquitination of histone H2A is required for methylation of histone H3, which relieves transcriptional represion in that locus (Kim et al., 2008). This intricate connectivity of diferent regulatory modes makes the eukaryotic system able to respond to the finest changes in its intra- and extra-celular environments. Recent studies reveal the interplay betwen ubiquitination and transcription in regulating various developmental aspects of C. elegans development. 31 1.4.1 Interplay during the development of an early embryo of C. elegans Spatial asymmetry in the early embryo of C. elegans is generated through interplay betwen ubiquitination and transcription. The single-celed C. elegans zygote divides asymmetricaly to produce two blastomeres, each with distinct developmental potential. SKN-1, a transcription factor required for mesoendoderm specification, is one of the first proteins to be asymmetricaly localized in the embryo (Bowerman et al., 1993; Bowerman et al., 1992). The protein acumulates in the posterior cel at 2-cel stage but not in the anterior sister cel. This asymmetry becomes more pronounced at the 4-cel stage. The protein is completely degraded from the embryo as it reaches the 8-cel stage (Bowerman et al., 1993). SKN-1 regulates the expresion of mesoderm-determining genes like med-1 in the anterior EMS blastomere (Maduro et al., 2001; Tenlen et al., 2006). efl-1, a transcription factor analogous to mamalian E2F, indirectly controls transcription of SKN-1. A HECT domain containing ubiquitin-ligase, EL-1, targets SKN-1 for degradation in the posterior cel at 2-cel stage, thus controlling its persistence in very narow spatial window. But, EFL-1 and EL-1 together regulate the spatial and temporal expresion patern of SKN-1. Deletion of both of these factors eliminates SKN- 1 asymmetry at the 2 and 4-cel stages, and the protein can be detected until the 28-cel stage (Page et al., 2007). 1.4.2 Interplay during sex determination of C. elegans The sex determination program is one of the pathways wel-studied at the molecular level. It integrates the ubiquitin-mediated regulation at TRA-1, the global 32 transcription regulator of sex-specific diferentiation. C. elegans develops either as X hermaphrodite or as XO male (Madl and Herman, 1979). The X-to-autosome (X:A) ratio determines the expresion levels of transcription regulators such as sex-1 (a nuclear hormone receptor) and ceh-39 (a ONECUT homeodomain protein), which are encoded on the X chromosome (Gladden and Meyer, 2007), and a T-box transcription factor sea-1 encoded on an autosome (Powel et al., 2005). These transcription factors together regulate the expresion of the gene xol-1 (XO lethal) (Figure 1-5), which integrates two downstream pathways: dosage compensation and sex determination (Luz et al., 2003). xol-1 in-turn regulates the complex of transcription factors SDC (sdc-1, sdc-2, sdc-3), esential to repres her-1 expresion in hermaphrodites (Pery et al., 1993; Zarkower, 2006). The her-1 gene encodes a secreted protein, which is a primary sex- determining signal. It promotes male development by inhibiting the function of a transmembrane protein TRA-2 (Hunter and Wood, 1992). TRA-2, together with proteins FEM-1, 2 and 3, regulate the activity of TRA-1, the terminal transcription factor in the sex-determination cascade. TRA-1 then brings about sex-specific diferentiation through downstream targets as discussed in the previous section (1.2.2). TRA-1 promotes female fate by suppresing transcription of male-specific genes in the hermaphrodite. TRA-1 is expresed in both the sexes and is primarily localized in the nucleus (Segal et al., 2001). But the overal TRA-1 levels are higher in hermaphrodites compared to males, implying that TRA-1 is regulated at the post- translational level (Schvarzstein and Spence, 2006). Recent studies report that TRA-1 protein is downregulated by a CUL-2-based E3 complex via ubiquitin-mediated 33 Figure 1-5: Sex-specific regulation of xol-1. xol-1 integrates both X and autosomal components to determine sexual fate. The molecular diagram indicates that CEH-39 and SEX-1 (nuclear hormone receptor) act to repres xol-1, whereas SEA-1 (T-box protein) functions to activate xol-1. In X worms, X-encoded factors out-compete to inactivate xol-1, but in XO animals, auotosomal factors outcompete to activate xol-1. The high level of XOL-1 protein present in XO animals then induces the male fate whereas the lower level of XOL-1 in X animals permits the hermaphrodite fate (Adopted from Gladden et al., 2007). 34 proteolysis, and that the FEM proteins are in fact part of this E3 complex (Starostina et al., 2007). FEM-1 functions as the substrate-recognition subunit while FEM-2 and FEM- 3 function as cofactors for CUL-2 ubiquitin ligase activity. CUL-2 negatively regulates somatic TRA-1 levels in males and in L4-stage hermaphrodites to promote masculine fate in the soma and the germline, respectively (Starostina et al., 2007). FEM-1 and FEM-3 proteins themselves are also indicated to be proteolyticaly degraded through the ubiquitin conjugation pathway. sel-10 (also known as egl-41) encodes a F-box protein that directly interacts with FEM-1 and FEM-3, targeting them for proteolytic degradation (Jager et al., 2004). The sex-determination pathway is ilustrated in the following Figure 1-6. The sex-determination cascade is regulated at each level though various mechanisms like translational regulation of tra-2 and fem mRNAs, receptor-mediated signaling via her-1 and tra-2, and stability of TRA-2 and TRA-1 proteins. As one can notice, tra-2 is subjected to complex and intricate regulation at al possible levels. TRA-2 promotes female fate via TRA-1 activity. During L4 larval stage of hermaphrodite, TRA- 2 activity needs to be transiently suppresed to alow sperm development. This transient downregulation of TRA-2 depends on rpn-10, which encodes a ubiquitin-binding protein, and ufd-2, which encodes an E3 ubiquitin ligase (Shimada et al., 2006). Sexual fate of C. elegans is thus regulated by the interplay betwen transcription and ubiquitin-mediated regulation at multiple levels. 35 Figure 1-6: Ilustration of sex determination pathway components. A) HER-1 is present only in males. It binds the transmembrane receptor TRA-2. TRA-2 in turn interacts with the FEM complex, which together with CUL-2 ubiquitinates TRA- 1. The FEM proteins are also ubiquitnated by the F box protein SEL-10 (Modified from 36 Zarkower 2005). B) Left, in males, TRA-1 gets degraded due to polyubiquitination by FEM and CUL-2 complex. In the absence of TRA-1, genes promoting the male fate are expresed. Right, in hermaphrodites, TRA-1 degradation is inhibited; as a result, it can supres the male genes promoting hermaphrodite fate. 37 This disertation reports the study of two genes involved in regulation of gene function in the context of C. elegans development. The first gene, uba-1, encodes the E1 ubiquitin-activating enzyme, which is esential to initiate ubiquitination and hence to regulate protein turnover during C. elegans development. The second gene, spe-44, encodes a putative transcription factor that is required for the regulation of sperm-specific gene expresion in C. elegans. 38 Chapter 2 E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout C. elegans development 2.1 Abstract Poly-ubiquitination of target proteins typicaly marks them for destruction via the proteasome, and provides an esential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade and its activity is necesary for al subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the C. elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at diferent developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantialy reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progresion in the early embryo, a proces that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with aleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and is one of the first conditional mutations reported for the esential E1 enzyme in a metazoan animal model. 39 2.2 Introduction Post-translational modification of proteins performs a critical role in regulating protein activity, and ubiquitin-mediated proteolysis has emerged as the key player in the control of protein turnover. Ubiquitin, a highly conserved smal protein, is covalently atached to a target protein through an enzymatic cascade, and the asembly of a poly- ubiquitin chain typicaly specifies that protein for rapid degradation via the 26S proteasome (Hershko and Ciechanover, 1998). Ubiquitin-mediated proteolysis thus provides an ?off? switch for governing the spatial and temporal distribution of proteins that are no longer needed. This mode of regulation is esential for normal celular proceses (e.g., cel cycle progresion and diferentiation), and defects have been implicated in human diseases such as cancers and neurodegenerative disorders (Ardley and Robinson, 2004; Nakayama and Nakayama, 2006). Ubiquitination of target proteins can also regulate function by mechanisms other than proteasome-mediated degradation. Mono-ubiquitination serves a signal for endocytosis and traficking of various cel surface proteins, and is also implicated in histone and transcription factor regulation (Haglund and Dikic, 2005; Mukhopadhyay and Riezman, 2007; Schnel and Hicke, 2003). The asembly of poly-ubiquitin chains can occur at diferent lysines within ubiquitin, which promotes diferent outcomes for the labeled protein. Conjugation at lysine 48 typicaly leads to proteasomal degradation, while linkage through lysine 63 can modulate protein activities in proceses as diverse as nuclear localization, DNA repair, or inclusion formation in neurodegenerative diseases (Chiu et al., 2006; Getha et al., 2005; Lim et al., 2005). 40 A trio of enzymes mediates the atachment of ubiquitin to substrate protein: the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase (Hershko et al., 1983). Repeated cycles of ligation to the initial ubiquitin lead to poly-ubiquitination. Substrate specificity is confered by the selective binding of individual E3 ligases to one or a few target proteins (Jackson et al., 2000). Eukaryotes typicaly posses a single gene encoding the E1-activating enzyme, tens of E2- conjugating enzymes, and as many as several hundred E3 ligases. Some E3 ligases are themselves multi-subunit complexes, in which a substrate recognition subunit specifies the protein targeted for ubiquitination. In vivo roles for ubiquitination in organismal development have been determined primarily through the characterization of specific E3 ligases. In the nematode Caenorhabditis elegans, E3 ligases regulate proceses as diverse as sex determination, cel cycle progresion, and synaptic signaling (Burbea et al., 2002; Feng et al., 1999; Juo and Kaplan, 2004; Schaefer and Rongo, 2006; Starostina et al., 2007). Studies of E2 conjugating enzymes indicate interactions with multiple E3s, as their relative numbers would predict. For example, inactivation of ubc-2 produces a broader range of phenotypes than inactivation of its known E3 partner apc-11 (Frazier et al., 2004). One of the best-characterized functions for ubiquitination and proteasomal degradation in C. elegans is the coordination of early events of embryogenesis (Bowerman and Kurz, 2006). The anaphase-promoting complex (APC) is a multi-subunit E3 ligase that is esential for completion of meiosis imediately after fertilization of the oocyte by the sperm (Davis et al., 2002; Golden et al., 2000). Ubiquitin-mediated proteolysis also plays a role in the degradation of several proteins that are involved in 41 establishment of anterior-posterior (A-P) polarity in the early embryo. These proteins become asymmetricaly localized at the first cel division, and failure to degrade these components correlates with developmental defects such as changes in cel fate specification and embryonic lethality. Formation of the A-P axis and progresion of the embryonic cel cycle requires the activities of a clas of E3 complexes known as Cullin- RING ligases (Bosu and Kipreos, 2008; DeRenzo et al., 2003; Kemphues et al., 1986; Liu et al., 2004; Pintard et al., 2003; Sonnevile and Gonczy, 2004; Xu et al., 2003). Mutations in components of the APC also afect A-P polarity, possibly as a consequence of defects in meiosis (Rappleye et al., 2002; Shakes et al., 2003). The E1 ubiquitin-activating enzyme lies at the apex of the enzymatic cascade, and manipulation of its activity might provide a crucial entry point for identifying the myriad roles performed by ubiquitin during development. Temperature-sensitive aleles of E1 have been identified in mamalian cel lines as cel cycle mutations that exhibit reduced ubiquitination and degradation of substrate proteins (Ciechanover et al., 1984; Finley et al., 1984). Similarly, a temperature-sensitive alele of E1 in yeast dramaticaly reduces ubiquitin conjugation and also leads to cel cycle arest (Ghaboosi and Deshaies, 2007). Conditional aleles have also been isolated in Drosophila in a scren for suppresors of hid-induced apoptosis during eye development (Le et al., 2008). Detailed characterization demonstrated the complexity of ubiquitin regulation in this system. Whereas weak aleles of the E1-encoding Uba1 gene block apoptosis, strong aleles promote cel cycle arest and death. Furthermore, these pro-apototic aleles promote non- autonomous proliferation in adjacent cels via elevated levels of Notch signaling. 42 We report here the isolation of a temperature-sensitive mutation in the C. elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Prior results for RNAi of uba-1 reported maternal sterility and embryonic lethality, with defects in meiotic progresion (Jones et al., 2002; Kamath et al., 2003; Sonnichsen et al., 2005). The uba- 1(it129) mutation recapitulates these phenotypes and also reveals several novel functions, including roles in sperm fertility, body size, and sex-specific development. The uba- 1(it129) mutation reduces in vivo levels of ubiquitin conjugates and causes a delay in meiotic progresion in the early embryo, consistent with a reduction in E1 activity. The uba-1(it129) mutation also demonstrates synthetic lethal interactions with known components of the anaphase-promoting complex and, as such, provides a sensitized genetic background for identifying new in vivo functions for other components of the ubiquitin cascade. 2.3 Materials and methods 2.3.1 Genetics C. elegans strains were derived from the wild-type isolate N2 (Bristol) and contained one or more of the following mutations: uba-1(it129)IV, uba-1(ok1374)IV, dpy-20(e1282)IV, fem-1(hc17)IV, fem-3(q20)IV, him-5(e1490)V, mat-3(or180)II, fzy- 1(h1983)I, spe-26(it112), or chromosome IV deficiencies eDf19 or mDf7. A linked uba- 1(it129) dpy-20(e1282) double mutant strain was generated to facilitate discrimination of homozygous and heterozygous lines in some experiments. The integrated oma-1::GFP transgenic line was constructed by Reuyling Lin (Lin, 2003). Strains were maintained on nematode growth medium (NGM) plates seded with E. coli strain OP50. Age- 43 synchronized populations of embryos were obtained by sodium hypochlorite treatment of gravid hermaphrodites. Strains were maintained at 15?C and shifted to 25?C as indicated for phenotypic analysis. Genetic manipulations were caried out acording to Brenner (Brenner, 1974). 2.3.2 Microscopy Microscopy was performed with an Olympus BX51TF or Zeis Axio Imager equipped with Nomarski DIC objectives and appropriate filter sets for fluorescent imaging and cooled CD camera for image capture. Images were procesed using the AxioVision (release 4.6) package and prepared for publication using Adobe Photoshop CS v. 9.0.2. Intact animals were typicaly mounted on 2% agarose pads for imaging. Body length was measured from captured images using ImageJ software v. 1.38. 2.3.3 Sperm assays Sperm morphology was asesed by disection of gonads from adult hermaphrodites or males in SM medium (Shakes and Ward, 1989a). Nuclear DNA morphology was visualized by 4'-6-Diamidino-2-phenylindole (DAPI) staining of sperm from disected gonads. In vitro activation of male spermatids was by treatment with monensin on poly-lysine-coated slides (Shakes and Ward, 1989a). Motility and localization of hermaphrodite sperm were determined in intact animals by fixation and staining with DAPI, then counting the number of sperm nuclei in the spermathecae and uterus. Sperm transfer was ascertained by vital staining of males (Hil and L'Hernault, 2001) with the mitochondrial dye MitoTracker Red CMXRos (Molecular Probes), then mating to unstained hermaphrodites anesthetized with tricaine and tetramisole. After 12 44 or 24 h, fluorescently-labeled male sperm within the hermaphrodite reproductive tract were visualized by microscopy using rhodamine filters. Self-fertility of hermaphrodites was asesed by shifting individual L3 animals to 25?C and counting the entire brood size. Cross-fertility of males was determined by mating with individual wild-type hermaphrodites or fem-1(hc17) females, then counting the number of male and hermaphrodite progeny produced by each animal after mating. 2.3.4 Cloning and molecular analysis The uba-1(it129) mutation was localized to chromosome IV betwen elt-1 and dpy-20 by thre-factor crosses. Single nucleotide polymorphisms that afect restriction sites (snip-SNPs) were employed as physical mapping markers of individual uba-1(it129) dpy-20(e1282) recombinants with Hawaian strain CB4856 (Wicks et al., 2001). Deficiency mapping was performed by complementation testing in uba-1(it129)/Df heterozygous strains. RNAi of candidate genes was performed by feding (Timons and Fire, 1998) and asesed by phenocopy of F1 embryonic lethality for treated adult hermaphrodites and by defects in adult tail morphology for treated L3 males. Complementation of the uba-1(ok1374) deletion alele was determined by generating it129/ok1374 heterozygous animals and performing temperature-shift asays as described for phenotypic characterization. Transformation rescue (Melo et al., 1991) was obtained by germ line microinjection of a 6.0 kb genomic fragment of the wild-type uba-1 gene mixed with plasmid pRF4, which contains the dominant roller marker rol-6(su1006), and linearized N2 genomic DNA at concentrations of 1, 50, and 100 ?g/ml, respectively. Stable roller transgenic lines were generated from uba-1(it129) hermaphrodites 45 maintained at 15?C, then rescue of sperm-specific sterility and embryonic lethality was scored after shifting to 25?C. The molecular lesion of the uba-1(it129) alele was identified by PCR amplification of the 6.0 Kb uba-1 genomic interval from mutant worms followed by sequence determination. In situ hybridization for uba-1 germ line expresion was performed on disected gonads following fixation (Le and Schedl, 2005). Digoxigenin- labeled, single-stranded sense and antisense probes were generated from a 1 kb cDNA fragment by linear amplification acording to the manufacturer?s protocol (Roche, Indianapolis, IN). Following hybridization, probe detection was by colorimetric asay with alkaline phosphatase (AP) conjugated anti-digoxigenin antibodies and nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate. Western blot analysis was performed on soluble worm lysates from age- synchronized young adult hermaphrodites shifted to 25? as L2 larvae. Lysates were obtained by one freze-thaw cycle, homogenization, and centrifugation for 10 minutes at 10,000 RCF. Protein concentration of the soluble fraction was quantified by Bradford asay. 10 ?g samples were fractionated by SDS-PAGE and transfered to PVDF membranes. Ubiquitin-conjugated proteins were detected by mouse anti-ubiquitin monoclonal antibody (1?) followed by HRP-conjugated goat anti-mouse IgG polyclonal antibody (2?; both Stresgen, Ann Arbor, MI). Duplicate gels were stained with Gelcode Blue (Pierce, Rockford, IL) to visualize total protein. 46 2.4 Results 2.4.1 Phenotypic characterization The temperature-sensitive it129 alele was isolated by Diane Shakes and, on the basis of sperm sterility and larval lethality, was provisionaly designated as spe-32 (S. Ward, pers. comm.). We have determined that spe-32 is alelic to uba-1 (se below), the sole E1 ubiquitin-activating enzyme in C. elegans, and have adopted the later gene name for the sake of clarity. Our detailed characterization of uba-1(it129) demonstrates a number of phenotypes, some of which are sex-specific, in addition to those mentioned above. Diferent phenotypes are manifested at diferent developmental stages (summarized in Table 1). To facilitate characterization, temperature-shift experiments were performed with age-synchronized populations of uba-1(it129) hermaphrodites. Adults shifted to the restrictive temperature produce dead embryos, and the number is equal to the number of progeny produced by wild-type animals at this temperature (Figure 1A). Embryonic arest is heterogeneous, based on the variable morphology of the embryos and the broad range in the number of nuclei observed with DAPI staining (Figure 1B). Temperature shift at any stage of embryogenesis leads to normal hatching, but 100% of the resulting larvae die at the L2 stage (data not shown). Thus, the uba-1 gene product is esential for both embryonic and larval development. Larvae that are shifted to the restrictive temperature at the L3 stage exhibit normal 47 Table 1. Sumary of uba-1(it129) phenotypes Stage shifted Phenotype(s) Adult F1 embryonic lethality Paralysis (male only) Embryo Larval lethality L2/L3 larva Sperm-specific sterility Change in body size Tail defect (male only) Paralysis (male only) Table 2-1: Sumary of uba-1(it129) phenotypes Divers phenotypes manifested by the temperature sensitive alele of uba-1 based on the life stage of the temperature shift. Every life stage of the mutant worms is afected by the temperature shift. 48 somatic development. However, reproduction is adversely afected in the adult hermaphrodite. These sterile animals lay only unfertilized oocytes instead of embryos, but produce viable progeny when mated to wild type males, indicating that the sterility is sperm-specific (Figure 1C). Viability of these outcross progeny is high (96%), suggesting that oocyte development (which occurs subsequent to sperm production in the hermaphrodite) is largely unafected by the mutation. Detailed characterization of the spermatogenesis defect (described below) indicates that these hermaphrodites produce appropriate numbers of morphologicaly normal sperm, but that the sperm are incapable of fertilization. Al of the above phenotypes are fully recesive, as heterozygous hermaphrodites are indistinguishable from wild type. These phenotypes are largely though not completely rescued in uba-1(it129) homozygous animals at the permisive temperature. There is an increase in embryonic lethality as wel as a decrease in the number of embryos produced (Figure 1A), which indicates that the uba-1(it129) gene product is not fully functional at 15?C. In addition, temperature causes a smal but significant (p < 0.001 by Student?s t- test) diference in body size betwen wild-type and uba-1(it129) adults (Figure 1D). When reared at 15?C, uba-1(it129) hermaphrodites are 16% longer than wild type. The opposite phenotype is observed at 25?C, with the uba-1(it129) mutants being 16% shorter than the wild type adults. In the course of generating heterozygous strains for phenotypic characterization, we observed strong maternal efect rescue of the early developmental defects. Homozygous uba-1(it129) progeny derived from +/uba-1(it129) hermaphrodites reared at the restrictive temperature exhibited litle embryonic or larval lethality (Table 2). 49 Figure 2-1: Defects in uba-1 hermaphrodites. A) Number of viable and inviable progeny produced by wild type (WT) or uba-1(it129) hermaphrodites at 15?C or 25?C. Shown are mean values and standard deviations (N=6) of total progeny. B) DAPI staining of uba-1(it129) embryos from adults shifted to 25?C. C) Sperm-specific sterility. Number of viable progeny produced at 25?C by wild type or uba-1(it129) hermaphrodites, either unmated (self) or mated with wild-type males (cross). Shown are mean values and standard deviations (N=6) of progeny produced in 48 h. D) Body length. Mean body length and standard deviations (N=20) of age- synchronized adult hermaphrodites. 50 Table 2. Maternal and paternal rescue of lethality Rescue Hermaphrodite genotype Male genotype Lethality (predicted) Maternal uba-1(it129) / + none (self-fertile) 3.8% (25%) Paternal uba-1(it129) / uba-1(it129) uba-1(it129) / + 7.2% (50%) Data are from five (maternal) or six (paternal) hermaphrodites. Mean total progeny numbers with s.d. are 123 ? 11 (maternal) and 73 ? 35 (paternal). Table 2-2: Maternal and paternal rescue of lethality The lethality in the homozygous progeny is rescued by the wild type copy of the uba-1 gene from either parent. 51 Maternal rescue was not complete for al phenotypes; although the homozygous hermaphrodites developed normaly into adulthood, sperm-specific sterility was stil observed in these animals. We also tested for paternal rescue by mating uba-1(it129)/+ heterozygous males to uba-1(it129) homozygous hermaphrodites. Again, embryonic and larval lethality (though not sperm sterility) were largely rescued (Table 2). Because the presence of a single wild-type copy of uba-1 in either the hermaphrodite or male parent efectively suppreses embryonic and larval lethality in homozygous mutant progeny, it suggests that the maternal or paternal contribution of UBA-1 protein is sufficient to alow somatic development to proced normaly until adulthood. 2.4.2 Male-specific phenotypes To facilitate the phenotypic characterization of males, we constructed a uba- 1(it129) him-5(e1490) strain [the him-5(e1490) mutation produces males via nondisjunction of the X chromosome] (Hodgkin et al., 1979). Temperature-shift experiments were performed with age-synchronized populations, and the same phenotypes were observed in males as above: embryonic and larval lethality and a reduction in body size (data not shown). Sperm-specific sterility of mutant males was asesed by crosing to fem-1(hc17) hermaphrodites, which lack sperm but produce oocytes that can be fertilized by mating. Experiments described below indicate that mating was succesful but no cross-progeny were produced, demonstrating that male sperm are incapable of fertilization. Thus, the same aray of defects are produced by the uba-1(it129) mutation in males and hermaphrodites. 52 We also observed additional phenotypes in uba-1(it129) males. The most conspicuous phenotype in the adult was constitutive protraction of the spicules (Figure 2A, first vs. second panel). These structures are part of the reproductive apparatus of the male tail, and are normaly extended only during insertion into the vulva for sperm transfer. A defect in spicule retraction was apparent in adult males at both the permisive and restrictive temperatures. Constitutively protracted spicules were observed in approximately one-third of uba-1(it129) males reared at 15?C and nearly al of those reared at 25?C. In some cases the spicules, gubernaculum, and surrounding tisues were everted, suggestive of structural defects in the integrity of the male reproductive tract as wel. Additional abnormalities in the male copulatory apparatus were observed in animals reared at the restrictive temperature. The tail of the wild-type male posseses a cuticular fan containing nine pairs of sensory rays (Figure 2A, first panel), which are involved in mate detection and the behavioral responses necesary for locating the hermaphrodite vulva. The size of the fan is greatly diminished in uba-1(it129) homozygous males raised at 25?C, which results in shortening of the tail tip and sensory rays as wel (Figure 2A, third panel). The number of rays is not afected, and other male reproductive structures appear superficialy normal by light microscopy. The shortened fan phenotype is semi-dominant: the fan expanse in heterozygous +/uba-1(it129) males is les than in wild-type but greater than in homozygous animals (data not shown). Therefore, proper formation of the male copulatory structures appears to be quite sensitive to the dosage of UBA-1 protein. 53 Figure 2-2: Defects in uba-1 males. A) Male tail defects. Shown are DIC photomicrographs of the male tail from wild type or uba-1(it129) animals grown at the indicated temperature. Bracket indicates the fan and sensory rays. Arow indicates the spicules. B) Reproductive succes of male mating. Graph indicates the mean number of male and hermaphrodite progeny produced by wild type hermaphrodites (N=6) mated to wild type or uba-1(it129) males grown at 15?C. Only the first 500 progeny were counted for wild type. Matings combined one hermaphrodite with five males, or thre hermaphrodites with 12 males (high #) for 24 h. Percent outcross is calculated by multiplying the number of male progeny by two, then dividing by the total number of progeny. C) Sperm transfer. Arow indicates fluorescently-labeled sperm from wild type or uba-1(it129) males localized within the spermatheca of unlabeled hermaphrodites after mating. D-F, male-specific paralysis. D) Young adult uba-1(it129) male. Arow indicates normal sinusoidal curve of tail. E) Older 54 adult uba-1(it129) male. Arow indicates flacid posture of tail. F) Dead uba-1(it129) male (arow) and two uba-1(it129) hermaphrodites. 55 The male tail structures are critical for mating behavior and sperm transfer, so aberations in the fan or in spicule function might adversely afect male reproductive succes. Sperm from wild-type males take precedence over hermaphrodite sperm such that only outcross progeny are produced until the male sperm are depleted, at which time the production of self progeny continues (Ward and Carel, 1979). Male sperm produce male and hermaphrodite progeny in equal numbers, while hermaphrodite sperm produce exclusively hermaphrodite progeny. Therefore, the number of outcross progeny, an indicator of male reproductive succes, can be readily calculated by determining the number of males produced. Reproductive succes was ascertained for homozygous uba-1(it129) males grown at the permisive temperature. Some of these animals have protruding spicules, which might be predicted to impair sperm transfer. The fertility of uba-1(it129) hermaphrodites at 15?C demonstrates that sperm function is normal at this temperature, so the production of outcross progeny was used as an indicator of succesful mating. Mating to wild-type males produced males and hermaphrodites in the expected 1:1 ratio, indicating that al of the offspring in the measured time interval resulted from fertilization by male sperm (Figure 2B, WT). In contrast, mating with uba-1(it129) males produced an average of only 56 male vs. 236 hermaphrodite progeny (Figure 2B, uba-1), suggesting that the number of outcross progeny is reduced. The same data could be explained if the nullo-X sperm, which produce male progeny, are les competent for fertilization than the X- bearing sperm that produce hermaphrodites. This explanation sems unlikely, because the percentage of male progeny is elevated if the density of males for mating is increased (Figure 2B, uba-1, high #). To eliminate conclusively this possibility, uba-1(it129) males 56 were mated to fem-1(hc17) adult hermaphrodites that lack sperm. Only outcross progeny are produced in this experiment and, although the numbers were low, males and hermaphrodites were observed in a ratio of 1:1 (data not shown). Therefore, the protruding spicule phenotype observed in uba-1(it129) males at the permisive temperature decreases the succesful transfer of sperm for fertilization. Reproductive succes was also characterized in the same manner for uba-1(it129) males shifted to the restrictive temperature at L3. No outcross progeny were observed from matings to either wild-type or fem-1(hc17) hermaphrodites. This failure might arise from the inability of sperm to fertilize the oocytes (as is true for hermaphrodite sperm at 25?C), or might be a consequence of the severe morphological defects in the male copulatory apparatus that occur at the restrictive temperature. A direct asesment of sperm transfer was performed to discriminate betwen the two possibilities. Males from him-5(e1490) strains that are wild-type or mutant for uba-1(it129) were raised at both 15?C and 25?C, stained with a fluorescent dye, then mated to fem-1(hc17) hermaphrodites that lacked sperm. Wild-type males reared at either temperature and mutant males reared at 15?C were succesful in mating 50-70% of the time, as revealed by the presence of labeled sperm in the fem-1(hc17) hermaphrodites (Figure 2C, first two panels). In contrast, uba-1(it129) males raised at 25?C succesfully transfered sperm in only two out of 10 instances. Although the eficiency of mating is reduced at 25?C, defects in the male copulatory structures arising from the uba-1(it129) mutation do not completely abrogate sperm transfer (Figure 2C, third panel). However, even those relatively rare succesful matings do not give rise to outcross progeny, indicating that uba-1(it129) sperm from males are incapable of fertilization at the restrictive temperature. 57 An additional, sex-specific phenotype was observed in uba-1(it129) males: a late onset, progresive paralysis in two-thirds of the animals (Figure 2, D-F). The paralysis initiates at the posterior of the male and proceds anteriorly as the worm ages, culminating in a completely paralyzed animal with a significantly shortened lifespan. Progresive paralysis is restricted to males, as uba-1(it129) hermaphrodites exhibit normal motility and lifespan (Figure 2F). The phenotype is not a consequence of aberant somatic development but instead occurs post-developmentaly, since delaying the temperature shift until adulthood stil results in paralysis. Therefore, the uba-1 gene product is required for the maintenance of neuromuscular function in the adult male. 2.4.3 Sperm-specific defect of uba-1 mutation Sperm development in C. elegans has been described in detail (Ward et al., 1981; Wolf et al., 1978), which alows the identification of specific cytological and functional defects in the developmental program that occur as a consequence of mutation. Normal spermatogenesis initiates from a mitoticaly dividing population of germ line stem cels. Primary spermatocytes separate from a syncytial cytoplasmic core and undergo a coordinated program of meiosis and diferentiation. The two meiotic divisions give rise to four haploid spermatids with highly condensed nuclei. These smal round cels separate from a larger residual body, which contains components not neded for subsequent steps in development. Activation by an extracelular signal converts the imotile spermatids into mature crawling spermatozoa capable of fertilization, and several compounds that promote activation in vitro have been identified (Nelson and Ward, 1980; Shakes and Ward, 1989a; Ward et al., 1983). Activation in hermaphrodites 58 occurs in the spermatheca, where the mature spermatozoa are stored. Activation of male spermatids occurs at the time of insemination, and the male spermatozoa crawl from the uterus into the spermatheca. Fertilization takes place within the spermatheca as the oocyte squeezes into this chamber of hermaphrodite reproductive tract, and the newly formed zygote then pases into the uterus. Most of the spermatozoa are dislodged and must crawl back into the spermatheca to await the next oocyte. Sperm-specific sterility caused by the uba-1(it129) mutation was characterized in greater detail, beginning with the early events leading to spermatid formation. DAPI staining of L4 and young adult hermaphrodites and males revealed no diferences in meiotic progresion, the number of sperm produced, or (for hermaphrodites) their initial localization to the spermathecae (Figure 3, A-B and data not shown). Activation of spermatids was normal in vivo and in vitro and produced crawling spermatozoa with no discernible defect in pseudopod movement or cel motility (Figure 3C). Since motility and localization appear normal and yet no zygotes are formed, the uba-1(it129) mutation produces mature spermatozoa that are nonetheles incapable of fertilization. A secondary defect in sperm function was detected later in adult hermaphrodites. Spermatozoa are displaced from the spermatheca into the uterus by each pasing oocyte, and must return to the spermatheca and await the next egg. Fertilization eficiency is esentialy 100% in wild type animals, with nearly every sperm being utilized for reproduction (Ward and Carel, 1979). Thus, the number of sperm in the spermatheca decreases in concordance with an increase in the number of progeny produced. Because uba-1(it129) spermatozoa are motile but incapable of fertilization, one might predict that numbers within the spermatheca would remain high throughout oocyte production. 59 Instead, the opposite phenomenon was observed, as sperm counts declined more rapidly in uba-1(it129) hermaphrodites than in wild type (Figure 3B). Furthermore, significant numbers of spermatozoa were detected in the uterus instead of the spermatheca (Figure 3A, uba-1). These cels are swept from the spermatheca by the unfertilized oocyte but are unable to return, and instead are expeled through the vulva when oocytes are deposited. Thus, although sperm motility and localization initialy appear normal, these proceses are clearly impaired in older animals. This observation may indicate a defect in the maintenance of sperm quality over time, which adversely impacts either motility or sperm-spermatheca interaction. 60 Figure 2-3: Sperm defects. A) Sperm localization in the hermaphrodite reproductive tract. Wild type and uba- 1(it129) adults at 25?C were fixed and stained with DAPI to count sperm nuclei. Arowheads, location of spermathecae; smal arows, sperm displaced into the uterus. B) Summary of sperm localization data. Shown are mean values and standard deviations per hermaphrodite (N=6). T0, before egg-laying commences; T1, after 1-2 ovulations; T2, 8 h post-T1; T3, 8 h post-T2. C) In vitro activation. Spermatids from wild type and uba- 1(it129) males at 25?C were activated with monensin. Arow indicates pseudopod of crawling spermatozoon. 61 2.4.4 Identification of it129 as uba-1 The identity of the it129 alele was determined through a combination of genetic and physical mapping strategies (Figure 4A). Thre-factor crosses placed this alele on chromosome IV betwen elt-1 and dpy-20 (F. Fel and S. Ward, pers. comm., and our own results; mapping data available at ww.ormbase.org). Single nucleotide polymorphisms that overlap restriction sites (snip-SNPs) were analyzed in recombinant lines (Wicks et al., 2001). Strains containing the deficiency eDf19 or mDf7 failed to complement it129, further limiting its position to the overlapping 310 kb interval. A total of 80 candidate genes within the interval were available from a large-scale RNAi feding library (Kamath et al., 2003). Al were tested for the ability to replicate two of the it129 phenotypes: F1 embryonic lethality of treated adult hermaphrodites, and tail deformation in adult males treated as larvae. Only one of the plasmids tested reproduced both phenotypes. That plasmid contains a fragment of the gene encoding the ubiquitin- activating enzyme E1, which in C. elegans is known as uba-1. Complementation tests confirmed that it129 is an alele of uba-1. The Gene Knockout Consortium (http:/celeganskoconsortium.omrf.org) has generated a deletion alele, uba-1(ok1374), that removes much of the third and fourth exons and is predicted to be a null mutation (Figure 4B). Mutants homozygous for uba-1(ok1374) exhibit embryonic lethality or early larval arest, so ok1374/it129 animals were obtained from crosses at the permisive temperature to alow recovery of viable lines. Complementation betwen the two aleles was tested by temperature shift at various developmental stages as described above. The identical phenotypes reported for it129 homozygotes were observed for ok1374/it129 double heterozygotes: embryonic lethality, larval lethality, 62 sperm-specific sterility, defects in male tail formation, and male-specific progresive paralysis (Figure 4C, and data not shown). Thus, ok1374 and it129 fail to complement each other and are both aleles of uba-1. The ok1374 deletion is a putative null alele, while the it129 mutation is probably hypomorphic (i.e., reduction of function; se Discussion). Therefore, we sought to ascertain whether it129/ok1374 heterozygotes were more adversely afected than it129 homozygotes. Most of the phenotypes observed in the it129 homozygous animals are highly penetrant, making enhancement dificult to detect. However, data from the complementation asay for sperm-specific sterility strongly suggest a more severe defect in it129/ok1374 animals. Cross-fertilization of sterile it129 homozygous hermaphrodites by wild type males yields progeny with high viability (96%; se Figure 1C). In contrast, cross-fertilization of sterile it129/ok1374 hermaphrodites produces embryos with very low viability (6%; Figure 4C). Furthermore, the same data demonstrate that the number of fertilized embryos is significantly lower for it129/ok1374 heterozygotes than it129 homozygotes (10 vs. 48, respectively). Sperm are normaly the limiting gamete for fertilization in C. elegans, but these results suggest that oocyte production might be defective in it129/ok1374 hermaphrodites. Therefore, we examined the gonads of these strains directly by DAPI staining. Germ cel development in C. elegans proceds distaly to proximaly within the gonad, and is most readily distinguishable by changes in nuclear morphology (Hirsh et al., 1976). In hermaphrodites, the proximal arm of the wild-type adult gonad contains a 63 Figure 2-4:Cloning and complementation. A) Schematic of cloning strategy. Shown at top is the interval of chromosome IV from elt-1 to dpy-20. Line two indicates the position of snip-SNPs identified in recombinant lines from N2 uba-1(it129) dpy-20(e1282) crossed with Hawaian strain CB4856. The next two lines indicate the endpoints and overlapping regions of chromosomal deficiencies eDf19 and mDf7, which failed to complement uba-1(it129). Eighty genes 64 within the 0.31 Mb overlap were screned by RNAi feding for two uba-1(it129) phenotypes: embryonic lethality and male tail defects. B) Predicted gene structure of uba-1. Shown are position of the Pro1024Ser misense mutation identified in the it129 alele and extent of the deleted region of the ok1374 alele. C) Complementation data for it129/ok1374 heterozygotes. Asay conditions for F1 embryonic lethality (N = 6 hermaphrodites), larval lethality (minimum 500 embryos), and sperm-specific sterility (N = 10 hermaphrodites) were identical to those used to characterize it129 homozyogotes; se Figure 1 and acompanying text. D) Germ line defects in it129/ok1374 heterozyogotes. Germ line nuclei were visualized by DAPI staining of fixed adult animals. The distal tip (DT) of the gonad is indicated for orientation. Top row shows a single gonad arm from (left to right) an it129 homozygous hermaphrodite, it129/ok1374 heterozygous hermaphrodite, it129 homozygous male, and it129/ok1374 heterozygous male. Bottom row shows a high-magnification image of the boxed region of the proximal gonad. Arows, oocyte nuclei in diakinesis. 65 row of individual oocytes whose nuclei are arested at diakinesis of meiosis I. Our analysis indicates that the germ lines of it129 homozygotes are similar to wild type hermaphrodites, and the proximal gonad contains morphologicaly normal oocytes whose six diakinetic bivalents are easily sen (Figure 4D, top and bottom left panels). In contrast, the germ lines of it129/ok1374 animals show an increased population of germ cels and a concommitant reduction in the number of oocytes in the proximal arm of the gonad. This defect in oogenesis is variable; some germ lines appear largely normal, while in other examples oocytes are absent and have been completely supplanted by an exces number of germ cels (as in Figure 4D, top and bottom second panels). A similar phenotype has been reported for mutations in a number of genes that govern the proliferation vs. meiosis decision, such as glp-1 (Bery et al., 1997). In addition, we also observed a spermatogenesis defect in the germ line of males. Wild-type adult males acumulate large numbers of highly condensed spermatid nuclei within the seminal vesicle. The it129 homozygous males likewise contain an abundance of compact spermatid nuclei (Figure 4D, top and bottom third panels). However, the seminal vesicle of it129/ok1374 males contain relatively few nuclei that also appear larger or les condensed than spermatid nuclei (Figure 4D, rightmost top and bottom panels). In both hermaphrodites and males, the mitotic and pachytene regions of the germ line in the distal gonad appear normal (albeit occasionaly reduced in size). These results suggest that the diferentiation of gametes in both sexes is impaired in the it129/ok1374 heterozygous mutants, but with opposite efects depending upon the type of gamete: males posses fewer spermatids than normal, while hermaphrodites contain an exces of germ cel nuclei rather than oocytes. Gamete-specific diferences in 66 proliferation and diferentiation have been reported previously. For example, loss of gld- 1 causes germ line overproliferation only in hermaphrodites undergoing oogenesis (Francis et al., 1995), while loss of puf-8 causes overproliferation only in sperm- producing germ lines (Subramanian et al., 2003). Transgene rescue of it129 with the wild type uba-1 gene further confirmed its identification. Initial atempts at rescue by germ line microinjection indicated that worms might be exquisitely sensitive to the dosage of this gene. Control injections with the rol-6 marker (Melo et al., 1991) produced numerous F1 rolling progeny with stable transmision in subsequent generations. In contrast, coinjection of uba-1 with rol-6 at typical concentrations resulted in low brood sizes with very few F1 rollers and no stably transmiting lines, suggestive of transgene toxicity. To reduce the gene dosage, the concentration of uba-1 DNA was decreased relative to rol-6 and genomic N2 DNA was also included in the injections. At the lowest concentration tested, four of sixten stably transmiting lines exhibited partial rescue of both sperm-specific sterility and embryonic lethality at the restrictive temperature. Therefore, the wild type uba-1 transgene is able to complement the it129 mutation. Expresion of a uba-1::GFP reporter transgene has been reported in a variety of somatic tisues but not the germ line (McKay et al., 2004), although a functional role for UBA-1 in this tisue is indicated by the mutant phenotype. Transgenes are often silenced within the germ line, so in situ hybridization was employed to detect transcription of the endogenous uba-1 gene within the gonad. Abundant expresion was detected in germ cels that had initiated meiosis in wild type hermaphrodites (during sperm and oocyte production) and males (Figure 5). Signal intensity appeared to be higher during oocyte 67 production; this observation was confirmed by comparing fem-1(hc17) hermaphrodites, which make only oocytes, to fem-3(q23) hermaphrodites, which make only sperm. Peak expresion occurs at pachytene of the first meiotic division, is absent imediately afterwards, and is detected again in late oogenesis. This patern is more apparent in the fem-1(hc17) gonad, which is from an older adult than the wild type hermaphrodite. Sequence determination of the uba-1 coding region from the it129-bearing strain revealed the molecular lesion. A single nucleotide substitution was detected that converts the proline at position 1024 to serine (Pro1024Ser, Figure 4B). The complete structure of E1 ubiquitin-activating enzyme has not yet been determined, but X-ray crystal structures of the activating enzymes for ubiquitin-like proteins SUMO and NED8 are available (Lois and Lima, 2005; Walden et al., 2003). The proline residue that is mutated in uba- 1(it129) maps near the active site where the ubiquitin moiety is predicted to be covalently atached to the E1 protein. On the basis of the structural data, the Pro1024Ser mutation might be expected to alter catalytic activity of the enzyme. 68 Figure 2-5: In situ hybridization of gonads. Panels on the left show the uba-1 expresion patern in disected gonads. Panels on the right show nuclear morphology by DAPI staining. From top to bottom, gonads are from adult hermaphrodites during oogenesis, adult males during spermatogenesis, fem-1(hc17) adult hermaphrodites that produce only oocytes, and fem-3(q23) adult hermaphrodites that make only sperm. At least 20 gonads were examined for each genotype or sex. DT, distal tip of gonad; P, pachytene region. 69 2.4.5 In vivo defects in ubiquitination and embryogenesis The uba-1 gene encodes the only known E1 ubiquitin-activating enzyme in C. elegans, so a defect in its activity is predicted to impair subsequent steps in the enzymatic cascade and cause an overal decrease in the level of ubiquitination on substrate proteins. We tested this hypothesis directly by using ubiquitin-specific antibodies to ases the amount of ubiquitination in worm protein lysates. To control for variations in ubiquitination activity at diferent stages of development, we extracted protein from age- synchronized young adult hermaphrodites shifted as L3 larvae. Since these uba-1(it129) animals are infertile due to sperm-specific sterility, we included as an additional control a strain containing spe-26(it112) (a temperature-sensitive, sperm-specific sterile mutation). Western blots show a significant reduction in the amount of ubiquitin signal in uba- 1(it129) protein extracts compared to wild-type and spe-26(it112) controls (Figure 6). Note that the level of ubiquitin in the high molecular weight region of the blot is particularly diminished, presumably reflecting a substantial reduction in the amount of poly-ubiquitinated substrates. Therefore, the uba-1(it129) mutation exhibits an in vivo decrease in protein ubiquitination. Reduced ubiquitination is predicted to adversely impact proteasomal degradation of target proteins. Wel-characterized roles for ubiquitin-mediated proteolysis in C. elegans occur during the early events of embryogenesis. The anaphase-promoting complex (APC) is an E3 ligase that is required for degradation of the meiotic inhibitor securin (Kitagawa et al., 2002). Complete loss of APC activity results in metaphase arest of the one-celed embryo (Golden et al., 2000). 70 Figure 2-6: Western blot for ubiquitin. Panel on the left (anti-Ub) shows the overal level of ubiquitin conjugates from wild-type, spe-26(it112), or uba-1(it129) young adult hermaphrodites. Equal amounts of soluble protein extracts were detected with ubiquitin-specific monoclonal antibody. Panel on the right (Coomasie) shows the same extracts stained for total protein. Size standards (MW) are indicated to the far right. 71 The uba-1(it129) mutation does not produce the one-celed arest caused by loss of APC activity, but instead miics the multicelular embryonic lethality resulting from reduced APC function. This phenotype is produced by hypomorphic mutations in APC components or by synthetic interactions betwen some pairs of temperature-sensitive aleles (i.e., each single mutation has no efect at the permisive temperature, whereas the combination of both mutations causes maternal embryonic lethality) (Shakes et al., 2003). Since UBA-1 and APC function in the same enzymatic cascade, mutations in both might likewise exhibit a synthetic interaction. Therefore, we tested the uba-1(it129) alele in combination with APC components. Double mutants of uba-1(it129) with either the APC subunit mat-3(or180) (Golden et al., 2000) or the APC activator fzy-1(h1983) (Kitagawa et al., 2002) resulted in maternal embryonic lethality at the permisive temperature (Table 3). Early embryogenesis was examined in uba-1(it129) adult hermaphrodites shifted to 25? for defects in meiotic progresion or A-P polarity in the first cel division. An oma- 1::GFP transgene was used to alow visualization of embryonic polarity (Lin, 2003). In wild-type hermaphrodites, OMA-1::GFP protein is evenly distributed throughout the cytosol and excluded from the intact pronuclei of the one-celed embryo. Our observations at 25?C indicate that the protein is also concentrated on the sperm centrioles and mitotic spindle. Ubiquitin-mediated proteolysis at the first cel division degrades the bulk of OMA-1::GFP. The protein is absent in the anterior (A) cel of the two-celed 72 Table 3. Synthetic interactions betwen double mutants Genotype + mat-3(or180) fzy-1(h1983) + WT WT WT uba-1(it129) WT Mel Mel WT, wild type; Mel, maternal embryonic lethality. Data are from a minimum of ten hermaphrodites for each genotype reared at the permissive temperature of 15?C. Table 2-3: Synthetic interactions betwen double mutants Worms double heterozygous for uba-1 and weak aleles of APC mutants show synthetic maternal embryonic lethality at permisive temperature. 73 embryo, and the remaining OMA-1::GFP becomes asociated with P granules in the posterior (P) cel. In uba-1(it129) animals, the patern of OMA-1::GFP in one-celed embryos is indistinguishable from wild-type. OMA-1::GFP degradation during the first cel division is likewise identical, and the protein persists only in the P cel. However, progresion of the zygote through the first division is slower than normal for uba-1(it129) embryos. The delayed progresion leads to an increase in the number of one-celed embryos within the uterus, which is easily visualized by the presence of OMA-1::GFP (Figure 7A). Wild-type hermaphrodites typicaly contain a single one-celed embryo in each arm of the gonad; in contrast, uba-1(it129) mutants posses an average of thre one- celed embryos per gonad arm (Figure 7B). In addition, 35% of the uba-1(it129) hermaphrodites contained a crushed zygote within the uterus. Formation of the rigid eggshel is completed late in meiosis, so these crushed zygotes might be either an indirect consequence of the observed meiotic delay or indicate a structural requirement for ubiquitination in the embryo imediately following fertilization. The delay in progresion through the first embryonic division was examined in greater detail. Upon fertilization, the oocyte nucleus completes the first and second meiotic divisions. The oocyte and sperm pronuclei met and fuse, then undergo the first mitotic division. The percentage of embryos observed at each of these stages (meiosis, pronuclear migration and fusion, and mitosis) was determined for wild-type and uba- 1(it129) animals. The fraction of one-celed embryos in the meiotic and pronuclear stages was equivalent in wild type, but approximately two-fold higher in the meiotic stage for uba-1(it129) embryos (Figure 7C), suggesting that meiosis is acutely sensitive to UBA-1. 74 Figure 2-7: OMA-1:GFP expresion. A) Adult hermaphrodites expresing the oma-1::GFP integrated transgene. Shown are examples of wild type and uba-1(it129) animals that contain one and four one-celed embryos, respectively. B) Frequency of one-celed embryos in the uterus. The number of one-celed embryos per gonad arm were counted for wild type (N=40) and uba-1(it129) (N=34) hermaphrodites. C) Distribution of one-celed embryos in the meiotic, pronuclear, and mitotic stages of development, as visualized by OMA-1::GFP. 75 Despite this significantly skewed distribution (p < 0.001 by Pearson?s chi-square test), there were no gross defects in nuclear or celular morphology or OMA-1::GFP distribution as embryonic development progresed. 2.5 Discusion We report here the isolation and characterization of a temperature-sensitive mutation of the uba-1 gene, which encodes the E1 ubiquitin-activating enzyme of C. elegans. Activation by E1 is the first step in the enzymatic pathway that leads to the conjugation of ubiquitin to target proteins. Manipulation of E1 activity by temperature shift provides a mechanism for identifying the many roles for ubiquitination throughout development. Efects of the uba-1(it129) mutation are manifested at both the organismal (i.e., embryonic and larval lethality, reduction in body size) and celular (sperm-specific sterility) levels, and also result in sex-specific diferences of developmental (formation of the male copulatory apparatus) and post-developmental (late-onset male paralysis) proceses. The uba-1(it129) mutation causes a substantial reduction of in vivo levels of ubiquitin-conjugated substrates, exhibits synthetic embryonic lethality with components of the anaphase promoting complex (an E3 ubiquitin ligase), and produces delays in early embryonic events known to be regulated by ubiquitin-mediated proteolysis. Taken together, the data indicate that the uba-1(it129) mutation results in a temperature-sensitive reduction in its ubiquitin-activating enzymatic activity. Since the uba-1 gene product is the only E1 enzyme in C. elegans, a reduction in its activity is predicted to negatively impact the function of E2 and E3 enzymes globaly. This reduction would extend the half-life of proteins normaly targeted for the proteasome, as 76 wel as altering the localization and/or activities of other ubiquitin-conjugated substrates. Some of these downstream pathways wil be more or les sensitive to a reduction in E1 activity, but the result wil be a decrease in the rate of ubiquitination for a wide variety of substrate proteins. In support of this model, Western blotting with anti-ubiquitin antibodies demonstrated an overal reduction in ubiquitin labeling of extracts from the uba-1(it129) mutant strain (Figure 6). Also, structural data from related E1 enzymes predict that the Pro1024Ser mutation in uba-1(it129) might alter its catalytic activity. Finaly, the model is consistent with our results in the candidate gene scren (in which reduced levels of UBA-1 by RNAi reproduced both the embryonic lethality and male tail defects) as wel as the phenocopy of APC hypomorphic aleles rather than strong loss-of- function mutations (i.e., multicelular vs. one-celed embryonic arest). An alternative hypothesis, that the uba-1(it129) mutation blocks only one or a few E2/E3 pathways, is les likely. The observed reduction of in vivo ubiquitination in the mutant would require that the bulk of ubiquitin conjugation be mediated by one or a few E3 ligases; however, the hundreds of E3s that are present in C. elegans argue against this model. Furthermore, the range of phenotypes produced by the uba-1 mutation is much broader than those reported for inactivation of any single E2 or E3 enzyme (Kipreos ET, 2005), consistent with its participation in multiple E2/E3 pathways. We clearly demonstrate genetic interactions betwen uba-1(it129) and one E3 pathway, the APC, via synthetic embryonic lethality with mat-3 or fzy-1 aleles. However, the sperm-specific fertilization defect appears to involve a diferent E3 pathway. This phenotype is not observed in APC mutants but has been reported for mutations in spe-16, which has 77 recently been determined to encode an E3 ubiquitin ligase homolog (Steve L?Hernault, personal communication). Some efects of the uba-1 mutation can be interpreted in light of the variety of phenotypes that arise from the loss of individual E2 or E3 activities. For example, embryonic and larval lethality have been reported for a number of E2 and E3 homologs in large-scale RNAi screns (Jones et al., 2002; Kamath et al., 2003; Sonnichsen et al., 2005). However, the majority of these genes have not been further characterized and, absent additional knowledge of which proteins are substrates for particular E2 and E3 enzymes, it?s dificult to speculate on the molecular mechanisms responsible for the observed lethality. In other instances, the uba-1 mutant phenotype suggests a previously unidentified role for ubiquitination. Body size in C. elegans is governed by a canonical TGF-? signal transduction pathway that initiates with the DBL-1 ligand (Savage et al., 1996; Suzuki et al., 1999). Components of the TGF-? pathway in other organisms are known to be regulated by ubiquitin conjugation (Itoh and ten Dijke, 2007). Diferent ubiquitin modifications produce antagonistic efects on signal transduction: mono-ubiquitination of Co-Smad stabilizes the protein and promotes signaling, while poly-ubiquitination of R- Smad leads to its proteasomal degradation and down-regulation of signaling. Given the efects of the uba-1 mutation on C. elegans body size, it sems likely that components of the DBL-1/TFG-? pathway are similarly regulated by ubiquitin. The sperm-specific sterility of uba-1(it129), coupled with the recent identification of spe-16 as an E3 ubiquitin ligase homolog (Steve L?Hernault, personal communication), indicate a previously uncharacterized role for ubiquitin in C. elegans 78 spermatogenesis. Ubiquitination is known to be esential for sperm function in a wide variety of organisms, and roles in mamalian spermatogenesis include regulation of the meiotic cel cycle, histone modification and chromatin remodeling, protein sorting during sperm diferentiation, and quality control for defective sperm (Barends et al., 1999a; Guardavacaro et al., 2003; Morokuma et al., 2007; Sutovsky et al., 2001). In C. elegans, early events like meiosis appear unafected by the uba-1(it129) mutation, suggesting that the infertility of these morphologicaly normal spermatozoa is due to a later defect in sperm development. In a manner analogous to mamalian sperm, ubiquitination in C. elegans might function in protein sorting as the spermatids divide from the residual body. Erors in this proces are known to adversely afect sperm function: mutation of spe-15, which encodes a myosin homolog, impairs the asymetric segregation of proteins during spermatid budding and causes sperm-specific sterility (Keleher et al., 2000). Alternatively, ubiquitination might promote proteasomal degradation of a protein that inhibits fertilization, and decreased activity of UBA-1 would lead to inappropriate persistence of the proposed inhibitor. Spermatid activation and downstream events occur in the absence of new protein synthesis, so degradation of pre-existing component(s) is a plausible mechanism of regulation. Another possibility is that uba-1(it129) infertility might reflect a role for ubiquitin-mediated proteolysis in the sperm-oocyte interaction. Fertilization in ascidians is mediated by an extracelular enzyme from sperm that conjugates ubiquitin to a sperm receptor on the egg surface, leading to its degradation via the proteasome (Sawada et al., 2002). Ongoing analysis is designed to determine if one (or more) of these hypotheses is correct. 79 Multiple E3 ligases are involved in formation of the reproductive structures of the male tail, so the defects observed in uba-1 mutant males might arise from impairment of one or more known ubiquitination pathways. Mutation of mat-1, which encodes the CDC27 subunit of the APC, causes a diminution in the size of the fan and sensory rays similar to the defect produced by uba-1(it129) (Shakes et al., 2003). The heterochronic gene lin-41, which encodes a homolog of the RING finger subclas of E3 ligases, is also required for proper formation of the male tail. A decrease in LIN-41 function causes precocious retraction of the male tail so that the fan and rays are reduced or absent (Del Rio-Albrechtsen et al., 2006; Slack et al., 2000). The DBL-1/TGF-? pathway (mentioned above) that determines body size also plays a role in formation of the spicules (Baird and Elazar, 1999), and might be implicated in the protruding spicule phenotype of uba- 1(it129) males. The late-onset paralysis and asociated lethality produced by the uba-1(it129) mutation is unusual in two regards: it is sex-specific, afecting only males, and can be induced after al somatic development is complete. There are few reports of such post- developmental phenotypes for C. elegans, and this property suggests a defect in the maintenance of neuronal and/or muscle function rather than its establishment. Roles for ubiquitination in C. elegans neuromuscular activity have been reported previously. Multiple E2 conjugating enzymes have been implicated in polyglutamine protein aggregation in muscle (Howard et al., 2007). E3 ligase complexes that have been demonstrated to afect either muscle or neuronal function include CHN-1/UDF-2, APC, KEL-8/CUL-3, SCF/FSN-1/RPM-1, SCF/LIN-23, and SCF/SEL-10 (Ding et al., 2007; Hoppe et al., 2004; Juo and Kaplan, 2004; Liao et al., 2004; Mehta et al., 2004; Schaefer 80 and Rongo, 2006). However, the paralysis of uba-1(it129) males is distinct from the more subtle neuromuscular defects reported for other ubiquitin pathway components such as APC (decreased duration of forward movement) or KEL-8/CUL-3 (changes in nose touch response and spontaneous reversal frequency) (Juo and Kaplan, 2004; Schaefer and Rongo, 2006). Furthermore, functional roles for al of these enzymes have been demonstrated in hermaphrodites, so the sex-specific ubiquitination that is responsible for male paralysis remains to be elucidated. Why are male-specific proceses, including the fertility defect of the male gamete (i.e., sperm), so acutely sensitive to the level of UBA-1 activity? One intriguing possibility involves the recently discovered role for ubiquitin-mediated proteolysis in the sex determination pathway. The TRA-1 transcription factor is the critical regulator of somatic and germ line sex determination and acts primarily as an inhibitor of male sexual fate (Hodgkin, 1987). Thre FEM proteins negatively regulate TRA-1 activity and thereby promote male cel fates, including sperm development in hermaphrodites (Hansen and Pilgrim, 1999). Starostina et al. (Starostina et al., 2007) demonstrate that the FEM proteins form an E3 ubiquitin ligase complex with CUL-2 that binds to and promotes proteasome-dependent degradation of TRA-1. Impairment of UBA-1 function by mutation would be predicted to decrease activity of the FEM/CUL-2 E3 complex, leading to an increase in TRA-1 levels that would inhibit male developmental proceses. This weakly feminizing efect might act synergisticaly with one or more of the E3 pathways described above. If this hypothesis is correct, then some of the sex-specific defects of the uba-1 mutation might be suppresed by a decrease in TRA-1 activity. 81 The observation of synthetic embryonic lethality betwen uba-1(it129) and mutations in components of the APC suggests a powerful approach for identifying new functions for downstream components of the ubiquitin pathway. A number of E2 and E3 homologs exhibit detectable phenotypes in genome-scale RNAi screns, but the majority are indistinguishable from wild type (Jones et al., 2002; Kamath et al., 2003). One possible explanation is that many of these enzymes are functionaly redundant, and that the determination of their roles wil require inactivation of multiple E2s or E3s. Alternatively, in some instances the reduction of E2 or E3 levels by RNAi might be insufficient to disrupt function. However, the uba-1 mutation provides a sensitized genetic background for detecting decreased activity of downstream enzymes. Reanalysis by RNAi screning of the E2 and E3 homologs in the uba-1 mutant strain is likely to reveal novel functions for a number of those genes whose roles are currently unknown. ACKNOWLEDGMENTS We wish to thank Diane Shakes for isolating and Sam Ward for providing the uba-1(it129) alele, Michael Stitzel of Geraldine Seydoux?s lab for providing the oma- 1::GFP strain, the Caenorhabditis Genetics Center and C.elegans Gene Knockout Consortium for providing strains, Andy Golden for providing strains and suggesting the synthetic lethal experiment, Eugene Melamud for asistance with the structural prediction, Sara Hapip for asistance with the RNAi candidate scren, and members of the lab and the Baltimore/Washington worm community for fruitful discussions. 82 Chapter 3 spe-44, a putative transcription regulator of sperm gene expresion 3.1 Introduction Development of an organism is achieved through celular diferentiation into specialized cel types. How cels diferentiate and develop into specific cel types is a fundamental question in developmental biology, and an important wealth of work has already been done to understand this phenomenon (Lewis et. al., 1998). The cel type is determined by the specific set of genes that govern the proces towards the terminal diferentiation stage. The expresion of genes is precisely regulated in a spatial and temporal manner to restrict the ?mesage? pased on to the cel, which then gets determined to be of specific fate. As discussed in Chapter 1, transcriptional control is one of the modes for regulating gene expresion in a cel-specific context. Studying the transcriptional control of gene expresion can help understand how cels control a particular order of gene expresion in spatial and temporal context. Spermatogenesis in Caenorhabditis elegans provides a good model system to study stem cel diferentiation and cel fate specification. The germline in C. elegans changes its fate during hermaphrodite development. The proliferative germline initialy diferentiates into sperm, but later in life the fate switches to oocyte. The spermatogenic germline goes through series of changes at the single cel level that are morphologicaly dramatic yet at the same time esentialy similar to universal proceses such as asymmetric cel division and signal transduction. Studying the transcriptional regulation 83 of the genes necesary for sperm diferentiation and development can give insights about the fundamental mechanisms of celular diferentiation and development in a complex eukaryotic system. 3.1.1 Sperm development in C. elegans The proces of development of the haploid spermatozoon from undiferentiated germline nuclei is refered to as spermatogenesis (Figure 3-1). The germline nuclei divide meioticaly, generating four haploid spermatids plus a residual body that contains components not needed for subsequent steps of development. The residual body eventualy gets degraded or absorbed. The spermatid diferentiates from a single spherical cel into a motile spermatozoan through the proces of activation or spermiogenesis. The terminaly diferentiated spermatozoon in C. elegans is an asymmetric cel with an amoeboid pseudopod for motility instead of a flagelum (Ward et al., 1981).. Although sperm development is esentialy the same in both hermaphrodites and males, there are thre major diferences. First, spermatozoa in males are larger in size compared to hermaphrodite spermatozoa, which gives them a competitive advantage during mating (LaMunyon and Ward, 1998). Second, the male germline continues to diferentiate into spermatids, whereas the hermaphrodite germline switches to oogenesis at the onset of adulthood. The third diference is in the storage form and timing of activation for the spermatids (Figure 3-3). The spermatids are stored in the proximal region of the gonad in hermaphrodites and get activated after being transfered to the spermatheca. In males, spermatids are stored in the seminal vesicle and spermiogenesis 84 Figure 3-1: Sperm development in wild type worm. Schematic diagram of stages during sperm development in C. elegans. The proces of sperm development is refered to as spermatogenesis and the activation of spermatids into spermatozoa is caled spermiogenesis. (Modified from L?Hernault, 2005) 85 occurs in the hermaphrodite uterus after mating (Ward et al., 1983). During sperm development, germline diferentiation proceds from the distal to proximal region of the gonad, so that the spatial distribution reflects temporal stages of development (Figure 3-2). In the distal region of the gonad, the germline proliferates mitoticaly. It goes through a transition zone (Figure 3-2B) and enters the meiotic cycle (Figure 3-2C). Nuclei in distinct phases of meiotic prophase I can be observed with their characteristic chromosomal paterns. Crescent-shaped nuclei (Figure 3-2B) are transitioning from mitosis to meiosis, while chromosomes in pachytene stage form a ?bowl of spagheti? as shown in Figure 3-2C. Nuclei are held together in a cytoplasmic core caled the rachis until this point (Hirsh and Vanderslice, 1976). As the nuclei progres through meiosis, they start budding off from the rachis as primary spermatocytes. As the primary spermatocytes complete meiosis I, the 4N nuclei condense giving a characteristic patern of the paired file of nuclei (arows in Figure 3-2) in the proximal gonad. After meiosis I, primary spermtocytes can either enter meiosis I directly without going through cytokinesis or separate into two secondary spermatocytes, each with a 2N nucleus (Ward et al., 1981). Meiosis I produces 4 haploid nuclei from one primary spermatocyte or 2 haploid nuclei from a secondary spermatocyte. These haploid nuclei bud off in an asymmetric cytokinesis event as round spermatids from a central anucleate mas of cytoplasm caled the residual body (Figure 3-1). 86 Figure 3-2: Progresion of germline diferentiation in the gonad during spermatogenesis. (A) The distal tip of the gonad with mitotic nuclei progres further into meiotic cycle (C) at the gonad bent through the transition zone (B). The proximal region of the gonad ilustrated the characteristic patern of the primary spermatocyte nuclei in paired file. Spermatocytes complete the meiotic division budding off four haploid condensed sperm nuclei (D). 87 Terminaly diferentiated spermatids contain a haploid nucleus, many mitochondria, and fibrous body-membranous organeles (FB-MOs) described in detail in Ward et al., (1981) and Wolf et al., (1978). Components like ribosomes, tubulin and actin are left behind in the residual body, which is degraded or reabsorbed (Figure 3-1) (Machaca et al., 1996). The spermatids are stored in the spermathecae within proximal region of the gonad in hermaphrodites and in the seminal vesicle of males as shown in Figure 3-3. Spermiogenesis, activation of spermatids into motile spermatozoa, occurs after mating for male sperm (Ward et al., 1983) and after entering the spermathecae for hermaphrodite sperm. The entire proces of spermiogenesis is initiated and completed without any new protein synthesis. The in vivo signal for sperm activation is not yet known, but spermatid can be activated in vitro using chemical agents that elevate the pH of sperm cytoplasm, e.g. the ionophore monensin or the weak base triethanolamine (Argon and Ward, 1980; Ward et al., 1983). The pseudopod is esential for motility in C. elegans sperm. Motility is achieved by continuous polymerization and depolymerization of the major sperm protein (MSP) in the pseudopod region (Roberts and Ward, 1982; Ward and Klas, 1982). MSP is synthesized very early as the primary spermatocytes celularize and then MSP polymers are stored in the FB-MOs as spermatocytes mature into spermatids. FB-MOs and MSP go though dynamic spatial redistribution as the sperm cel diferentiates into the motile spermatozoon (details in Roberts et al., 1986). 88 Figure 3-3: Germline distribution and morphology of hermaphrodite and male gonad. Two armed gonad from hermaphrodite (top) and single armed male gonad (bottom) showing the distribution of germline. In hermaphrodites, spermatozoa are stored in spermathecae (sp) on each side of the uterus, while in males, spermatids are stored in the seminal vesicle in the proximal gonad (PG). DG: Distal gonad. Image adopted from Schedl et al., (1997). 89 The pseudopod is also required to maintain adherence to the spermathecal wal (Shakes and Ward, 1989; Ward and Carel, 1979). As the mature oocyte enters the spermatheca, the spermatozoa release themselves from the wal and one of them fertilizes the oocyte (Ward and Carel, 1979). The sperm are swept out of the spermatheca as the egg pases through the spermatheca and they need to crawl back with the help of pseudopod to position themselves again in the spermathecal wal invasions. 3.1.2 Mutational analysis of spermatogenesis in C. elegans The hermaphroditic system of reproduction in C. elegans makes it easy to isolate and study spermatogenesis-defective (Spe) mutations. The Spe mutation can be maintained by mating sperm-sterile hermaphrodites to wild type males. The combination of genetics of C. elegans and cytological analysis of sperm development offers an easy tool to track the functional role of these Spe mutations in spermatogenesis. Microaray analysis has been used to identify the genes expresed in fem-1(h17) hermaphrodites, which produce only oocytes, and fem-3(q20) hermaphrodites, which produce only sperm. Comparison of these two expresion sets revealed 1343 genes enriched during spermatogenesis (Reinke et al., 2004, described in detail in the result section). Approximately sixty of these genes have been geneticaly characterized within the last two decades, but only half of them have been identified at the molecular level. These mutations are generaly categorized in thre types (Figure 3-4). The spe-8 clas afects spermiogenesis in hermaphrodites only ans comprises of 4 genes. The spe-9 clas, which afects sperm-oocyte interaction and thereby fertilization, comprises seven genes 90 and most of them encode transmembrane proteins (Chaterje et al., 2005; Kroft et al., 2005; Singson et al., 1999; Xu and Sternberg, 2003). The third clas of mutations afects various stages of the development and leads to aberant sperm. This clas has helped to elucidate the spermatogenesis pathway based on their arest points as explained in Figure 3-4. A few mutations like cpb-1 and we-1.3 afect more general functions like translation or cel cycle control and arest spermatogenesis during early stages of development (Lamitina and L'Hernault, 2002; Luitjens et al., 2000). Mutations that afect asymmetric distribution of components like spe-26 and spe-15 cause developmental arest before completion of meiosis (L'Hernault et al., 1988; Varkey et al., 1995). One subset of this clas produces ?terminal spermatocyte? arest. These mutations impair FB-MO morphogenesis (e.g. spe-4, spe-5) afecting either spermiogenesis or maturation into spermatids (L'Hernault and Arduengo, 1992; P. Hartley and S.W. L?Hernault, unpublished results). This subclas is discussed in more detail in the discussion section. 91 Figure 3-4: Schematic representation of mutations at their respective arrest points during spermatogeneis. The diagram is taken from L?Hernault 2005. It shows the spatial positioning where the specified gene product is required based on their mutational analysis. 92 3.1.3 Putative transcriptional regulators expressed during spermatogenesis Gene ontology annotation for the genes from the microaray analysis of germline- enriched genes (Reinke et. al., 2004) revealed about 4% of the sperm-enriched genes with predicted nucleic acid-binding domain. Only ten of those show significant homology to known DNA-binding domains (Table 3-1). These ten are important candidates to study gene expresion regulation during spermatogenesis. One of these ten is an established GATA transcription factor (Gileard and McGhee, 2001; Page et al., 1997). elt-1, which encodes this GATA transcription factor, is wel studied for its role in specifying hypodermal cel fate during embryogenesis (Smith et al., 2005). Mutant phenotypes during embryogenesis and further development have been characterized using genetic mutations and RNAi studies. The microaray experiment revealed up-regulation of this gene in during spermatogenesis, suggesting a novel function for ELT-1 during sperm development. ELT-1?s role as a transcription regulator was confirmed when it was identified as a DNA-binding factor for a sperm- specific promoter sequence in yeast-one hybrid analysis (Smith HE, unpublished). In situ hybridization confirmed its expresion in the spermatogenic germline (refer to Appendices). We requested deletion aleles for the remaining nine putative transcription factors from C. elegans Knockout Consortium. The deletion alele for C25G4.4 was the first one to be available for study. This chapter wil present genetic and cytological analysis of C25G4.4. The 93 Gene Homology Domain Identity ceh-1 Slouch homeodomain 81% elt-1 GATA binding protein GATA3 zinc finger 44% nhr-43 retinoic acid receptor RG1 zinc finger 26% C17H12.9 hepatocyte nuclear factor HNF6 homeodomain 67% C25G4.4 glucocorticoid modulatory element binding GMEB SAND domain 37% C44F1.2 glucocorticoid modulatory element binding GMEB SAND domain 41% F44D6.2 PRK1 asociated protein AWP1 zinc finger 36% F56F3.4 PRK1 asociated protein AP1 zinc finger 38% F26F4.8 shavenbaby-ovo zinc finger 28% T20H4.2 Kruppel asociated box KRAB zinc finger 30% Table 3-1: Putative sperm-gene regulators in C. elegans genome Predicted transcription factors form the genome which show diferential expresion in the spermatogenic germline of C. elegans. 94 predicted protein sequence of C25G4.4 contains a SAND domain, a recently identified DNA-binding domain (Bottomley et al., 2001). Acording to the microaray data, it is expresed at two-fold higher level in spermatogenic germline than in oogenic germline (Reinke et al., 2000). In situ hybridization revealed specific spatial and temporal expresion of the gene imediately prior to sperm production. Genetic analysis of a deletion alele of C25G4.4 has revealed its functional role during sperm development as indicated by the Spe phenotype, hence now it is caled spe-44. Cytological analysis of this deletion alele of C25G4.4 showed a ?terminal spermatocyte? phenotype similar to the one observed in spe-4, spe-5, spe-26 and spe-39 mutations. Each of these genes has a distinct function in the spermatogenesis pathway, yet they produce the same mutant phenotype. If C25G4.4 is necesary for the expresion of one or more of these genes and loss of C25G4.4 would lead to loss of expresion of the downstream gene(s). This could be a possible explanation for the similar phenotype observed in these mutants. Thus, these genes could be the downstream targets of the putative transcription factor encoded by C25G4.4, functioning in the same regulatory cascade. The detailed analysis of the deletion alele of spe-44 using cel biological and genetic tools and its functional role during sperm development is presented in this chapter. 3.2 Materials and Methods 3.2.1 Strains C. elegans var. Bristol, N2 strain, was used as wild type. The strains used in this study were obtained from Caenorhabditis Genetics Center (CGC), unles otherwise mentioned. The aleles used were dpy-20(e1282)IV, fem-1(hc17)IV (Nelson, 1978), fem- 95 3(q20)IV, him-5(e1490)V (Hodgkin et. al., 1979). The deletion alele ok1400 of C25G4.4 was obtained from C. elegans Knockout Consortium (http:/celeganskoconsortium.omrf.org) as a heterozygous strain. The deletion strain has been asigned the name spe-44 following the CGC nomenclature standards. The spe- 44(ok1400) dpy-20(e1282)IV homozygous strain was created in the lab. Heterozygous strains of spe-44(ok1400)IV and spe-44(ok1400) dpy-20(e1282)IV over rearangement nT1 were created in the lab. This rearangement is a translocation of chromosome IV and V that also contains an integrated transgene qls51 with the gren fluorescent protein (GFP). Al strains were maintained on NGM plates seded with E. coli (OP50) at permisive temperature of 15 0 C unles otherwise mentioned. Genetic manipulations were caried out acording to Brenner (1974). 3.2.2 Single Worm PCR Individual heterozygous and homozygous spe-44(ok1400) and spe- 44(ok1400)dpy-20(e1282) hermaphrodites were isolated based on the sterility phenotype at adulthood. Multiplex single-worm PCR was performed on 6 replicate samples of each genotype using the primers as diagramed in Figure 3-5. For each reaction, a single worm was picked in a 2.5?l drop of worm lysis buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl 2 , 0.45% NP-40, 0.45% Twen 20, 0.01% gelatin and 60 ?g/ml proteinase K) in the cap of 0.5?l PCR tube. The tube was spun briefly and frozen at -80 0 C at least for 10 minutes. Before seting up the PCR, the 96 Primer 343 CA GTA TA CGT TGT GAC GAG Primer 502 GAT GA GCA TC ACA TA TT C Primer 364 TCA CG TT TAT TCG AT TG 2000bp amplicon 700bp amplicon 500bp amplicon Figure 3-5: Cartoon of the primers used to detect C25G4.4 deletion. Schematic diagram of the primers used to detect the deletion in spe-44(ok1400) strain compared to wild type worms. WT C25G4.4 1.5 kb deletion 97 worm was lysed at 65 0 C for 1 hr 15 minutes and then at 95 0 C for 15 minutes. After lysis, each tube was added with the multiplex PCR mix (with additional 1.5 mM gCl 2 ) with primers 343, 502 and 364. The PCR was run at 37 cycles of 94 0 C for 30 sec, 55 0 C for 30 sec, 72 0 C for 2 min with final extension of 3 minutes at 72 0 C. 3.2.3 Worm microscopy DIC microscopy was performed on staged worms and disected gonads. The samples were mounted on 2% agar pads in M9 buffer (22 mM KH 2 PO 4 ; 42 mM Na 2 HPO 4 , 85.5 mM NaCl, 1 mM MgSO 4 ). Nuclear morphology was visualized using DAPI stain. Worms were fixed in a series of 30%-65%-95% ethanol at 65 0 C followed by acetone and then incubated with 100ng/ml DAPI in 1X phosphate buffered saline (PBS) (1.8 mM KH 2 PO 4 ; 10 mM Na 2 HPO 4 ; 137 mM NaCl; 2.7 mM KCl, pH 7.4) in the dark for 20-30 minutes. Disected gonads were incubated in the dark with 100ng/ml DAPI in 1X PBT (PBS-Twen 20 0.1%) for 5-15 minutes. Images were taken with appropriate filters using Zies AxioCam HRc and procesed using software AxioVision Rel 4.6. 3.2.4 RNAi for C25G4.4 RNAi construct (TF1) was generated by cloning a 637bp region amplified from wild type genomic DNA into pPD129.36 betwen inverted T7 promoters using HinDII and XbaI sites. The primers used were CC AG CT ATG TC GT GA GAC GTG and GC TCT AGA TCG TAG AG TCG ATG TC. The clone was then transformed in E. coli strain HT115. Synchronized populations of N2 (wild type) hermaphrodites were grown until L2- L3. Sets of 20 hermaphrodites shifted to NGM + isopropyl-beta-D-thiogalactopyranoside 98 (IPTG) + ampicilin plates expresing spe-44 RNAi construct or control (pPD129.36 in HT115 strain) in triplicate. The worms were maintained on RNAi plates until late adulthood and then were examined for unfertilized oocytes or sterility. Secondary spe-44 RNAi was performed in the same manner on the F1 progeny from treated P0 hermaphrodites. 3.2.5 In situ hybridization fem-3(q20) and fem-1(hc17) worm populations were bleached and hatched on NGM plate overnight at 15 0 C. The hatched L1 larvae were shifted to NGM plates with food at 25 0 C until appropriate stages. fem-3(q20) hermaphrodites produce only sperm while fem-1(hc17) produce only oocytes at 25 0 C 3.2.5.1 Worm disections Synchronized populations of worm strains were obtained at L3, L4 and adult stages. The worms were collected in PBS with 0.25 mM leavamisole. 200-300 worms were disected with a 20-gauge needle to extrude the gonad arms and then collected in sterile 3ml glas culture tube with the conical bottom. 3.2.5.2 sDNA probe synthesis spe-44 cDNA was amplified (1.3 kb fragment) from the clone pHS589B using anti-sense primer HES-539 (ACG ATC TC TT CTC CGA AG) and sense primer HES-540 (CT TCT AT ATC ATC AT ATC CGC). Using this cDNA as a template, single-strand DNA was linearly amplified using either 3? anti-sense or 5? sense primer with a Digoxigenin (DIG) labeled mix acording to the manufacturer?s protocol (Roche). Amplified sDNA was run on a denaturing agarose gel to confirm the single band and 99 size. sDNA was then precipitated with 0.2M NaCl and ethanol and resuspended in 250?l hybridization buffer. The probe was boiled for 1hr and stored at ?20 0 C until use. The probe concentration was determined by dot-blotting on a nitrocelulose membrane along with a marker of known concentration. The probe was detected using colorimetric asay with alkaline phosphatase (AP) conjugated anti-DIG antibody and nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) caled NBT/BCIP substrate by comparing the dot intensity to the control fragment. Both the sense and anti-sense probes were at 4ng/?l concentration. Prior to use, each probe was diluted 1:2 with the hybridization bufer and boiled for 5 minutes. 3.2.5.3 Gonad fixation and hybridization This proces was caried out acording to the protocol by Min-Ho Le and Tim Schedl (http:/ww.ormbook.org/toc_wormethods.html) with minor modifications. After fixation, the gonads were treated with proteinase K for 1hr at the concentration of 100 ?g/ml in PBT. Hybridization with the DIG-labeled sDNA probe (150 pg/?l) was caried out at 48 0 C for 36 hrs. Subsequent washes were also caried out at 48 0 C. After blocking with bovine serum albumin (BSA), the DIG probe was detected by colorimetric asay of AP-conjugated anti-DIG antibody with NBT/BCIP substrate. Finaly, gonads were mounted on an agar pad in PBS with 100 ng/ml 4'-6-Diamidino-2-phenylindole (DAPI) and the images were taken with Zies AxioCam HRc and procesed using software AxioVision Rel 4.6. 100 3.2.6 Western analysis Homozygous and heterozygous spe-44(1400)dpy-20(e1282) adult males were maintained on food without hermaphrodites for 24 hrs. 50 adult males of each genotype were washed with 1X PBS and resuspended in 20 ?l homogenization buffer with 1 ?l of 1M DT and 1 ?l of protease inhibitor mix. The pelet was stored frozen at -80 0 C. Imediately prior to SDS-PAGE, the pelet was boiled with equal volume of 2X sample buffer for 10 min. Equal volumes of the supernatants were loaded on the 10% SDS- PAGE gel after spinning at 6000 g for 10 minutes. After running, the gel was equilibrated with 1X transfer buffer (25 mM Tris-HCl, 0.2 M Glycine) without methanol. The nylon membrane was activated in 100% methanol, washed with water and equilibrated with 1X transfer buffer for 20 minutes. The gel and the membrane were sandwiched betwen 2 sheets of 3MM Whatman papers soaked in 1X transfer buffer. The entire asembly was sandwiched in a casete betwen 2 sponges soaked in 1X transfer buffer and the casete was imersed in an electroblot tank containing 1.5 liters of 1X transfer buffer keeping the membrane towards the anode side. The transfer was caried out at 100V for 60 minutes. After transfer, the membrane was blocked in 5% milk (nonfat powdered milk) in TBST (20 mM Tris pH 8, 150 mM NaCl, 0.05% Twen-20) for 1hr at room temperature. The membrane was washed in TBST for 3 times at 15 minutes interval. It was then incubated overnight at 4 0 C with polyclonal anti-MSP (a gift from Dr. David Grenstein, Kosinski et al., 2005) at 1:2000 dilution in 5% milk-TBST. The next morning, the membrane was washed and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Pierce) 1:25000 diluted in 5% milk- TBST for 1 hr at room temperature. The membrane was washed 3 times with TBST and 101 then developed using Supersignal substrate (Pierce). The membrane was exposed to X- ray film in the dark for 1 to 40 minutes. The film was developed using AFP imaging system. 3.2.7 Microinjection rescue Genomic region of 6449bp of spe-44 amplified from N2 genomic DNA was cloned in pHS584 using SOEing PCR (Horton et al.1990). The primers used were HES- 502+515 (GAT GA GCA TC ACA TA TT C and AA AG ATC CT CGT CTA AA AC TCT AT TA AG) and HES-364+516 (TCA CG TT TAT TCG GAT TG and AA CG C GTG AA TG TA TGA GT AA TAT ATA TT). Heterozygous spe-44(ok1400)dpy-20(e1282) worms were microinjected with a mixture of purified pHS584 and pRF4 plasmid with rol-6(su1006) at concentrations of 4 and 200 ?g/ml, respectively . Standard microinjection protocol (Melo et al., 1991) was followed. Stable roller transgenic lines were obtained by maintaining the injected worms at 15 0 C. The rescue of sterility was scored by maintaining individual L3 dumpy roler hermaphrodite from F4 generation to a separate plate. 102 3.3 Results Reinke et.al. (2004) performed microaray analysis to identify genes expresed specificaly during spermatogenesis or oogenesis by comparing transcriptional profiles in fem-3(q20)IV and fem-1(hc17)IV strains. These mutations are both temperature-sensitive: the gain-of-function alele q20 of fem-3 causes only sperm production in the hermaphrodite germline (Barton et al., 1987) and loss-of-function alele hc17 of fem-1 causes only oocyte production in the hermaphrodite germline (Nelson et al., 1978). Out of 18,010 total genes on the microaray, 4245 genes showed diferential expresion betwen fem-3(q20) and fem-1(hc17) with 2 to 71 fold diference in the expresion levels. Out of those genes, 1343 were overexpresed in fem-3(q20) compared to fem-1(hc17) and 702 showed significant enrichment in spermatogenic germline over oogenic germline (http:/wormgermline.yale.edu). Out of these 702 genes, 10 showed significant homology to reported DNA-binding domains based on the BLAST searches of NCBI nonredundant protein database, indicating their potential role in regulating gene expresion specificaly during sperm development. C25G4.4 is one of the genes that shows upregulation (2.215 fold) in fem- 3(q20) worms compared to fem-1(hc17) and also 3.7 fold upregulation in wild type worms at L4 stage (when the worm germline is spermatogenic) compared to glp-4(bn2) which lack germ cels (Beanan and Strome, 1992). C25G4.4 is predicted to encode a 424- amino acid protein with a region from 65 to 150 th amino acid homologous to the SAND domain (Figure 3-6). The SAND domain is a recently identified DNA-binding domain with KDWK conserved motif esential for DNA binding (Botomley et. al., 2001). Strong 103 homology (37% identity to the SAND domain in GMEB protein with e-value of 1.2e-12) to this DNA-binding domain and overexpresion in sperm-producing worms suggest that C25G4.4 is a putative regulator of sperm gene expresion. 3.3.1 C25G4.4 RNAi produces no phenotype To determine if C25G4.4 indeed plays a role during sperm development, RNAi was performed on wild type L2 larvae by feding at 15 0 C. Individual worms were maintained until late adulthood on the RNAi plates expresing C25G4.4 dsRNA or control RNAi with vector alone. Adult hermaphrodites were scored for the presence of unfertilized oocytes on the plates. This experiment was performed in triplicates with 20 individual worms in each. In al of these triplicate runs, al the adult hermaphrodites laid healthy brood and in normal numbers. None of the hermaphrodites laid any unfertilized oocytes during the first 4 days of the progeny-laying period. In C. elegans, sperm-specific genes are particularly resistant to RNAi for unknown reasons. There are at least 11 genes on chromosome I known to play a functional role during spermatogenesis (L'Hernault et al., 1988). A large-scale RNAi feding scren for genes on chromosome I, including al 11 known SPE genes, did not show any sperm-specific sterility (Fraser et al., 2000). Because of similar resistance to RNAi, it is possible that C25G4.4 RNAi did not reveal any sperm-specific fertility defects. 104 Figure 3-6: Alignment of SAND domain from homologous proteins. Protein sequence alignment of pfam SAND domain with the SAND domain encoded in C25G4.4, DEAF-1 and GMEB proteins. The red box indicated the four most conserved residues, KDWK of the SAND domain. 105 3.3.2 Deletion in C25G4.4 causes sperm-specific sterility We requested a deletion alele of C25G4.4 from the C. elegans Gene Knockout Consortium. The alele ok1400 was created by the Knockout Consortium by trimethylpsoralen treatment with UV-crosslinking to induce deletion mutations and the mutation was identified by PCR screning using gene-specific primers (Barstead R.J. 2000). The alele contains a 1577bp deletion leaving only 150bp in the first exon and 71bp in the last exon and is predicted to encode a null mutation. The strain carying the ok1400 deletion segregated worms that lay only unfertilized oocytes. These sterile hermaphrodites were able to produce viable progeny after mating with wild type males, which confirmed that the sterility is sperm-specific. The sterile hermaphrodites were backcrossed to N2 males a total of 6 times, selecting for sterile hermaphrodites each generation to eliminate other mutations from the genome. Heterozygous hermaphrodites were indistinguishable from wild type and segregated 24.6% sterile hermaphrodites of the total progeny further indicating that the alele is recesive. Single worm PCR was performed on the sterile and fertile hermaphrodites with multiplex PCR using a primer set external to the deleted region and one primer within the deleted region as shown in Figure 3-5. When the wild type copy of C25G4.4 is present in the worm, the primer sets would amplify two products of 2kb and 700bp. In a multiplex PCR, the shorter amplicon takes precedence and the 2kb product is never observed. A heterozygous worm, carying a wild type copy and a deletion copy of C25G4.4 would give two products of 700bp and 500bp. A worm homozygous for the deletion would amplify only a 500bp product as shown in Figure 3-7. 106 In every reaction, the sterile hermaphrodites amplified only the 500bp product (Figure 3-7, Lane 1-6) demonstrating that these hermaphrodites were homozygous for the C25G4.4 deletion. Fertile hermaphrodites amplified either only 700bp (lane 7) or both 700 and 500bp products (lane 8-10) being either wild type or heterozygous for the deletion, respectively. Thus, sperm-specific sterility always segregated with homozygous deletion alele ok1400 and is tightly linked to the C25G4.4 locus. Therefore, C25G4.4 was provisionaly named spe-44 acording to the Caenorhabditis Genetics Center (CGC) nomenclature standards. 3.3.3 Balancer and marker linked strains for spe-44 For easy screning of sterile spe-44(ok1400) worms, the mutation was linked with the phenotypic marker, dpy-20(e1282). The strain spe-44(ok1400 dpy-20(e1282)IV was created by crossing sterile spe-44(ok1400) hermaphrodites with dpy-20(e1282) males and then screning for sterile, dumpy recombinants. Mating these recombinants to wild type yield heterozygous spe-44(ok1400) dpy-20(e1282) hermaphrodites that segregate one quarter sterile-dumpy progeny as expected. A balancer strain was created to maintain a population of homozygous sterile worms. spe-44(ok1400) was balanced over nT1, a translocation of chromosome IV and V, which has an integrated qls51 transgene. The transgene expreses GFP in the pharynx of the worms carying the nT1 chromosome. Since worms homozygous for the 107 Figure 3-7: ok1400 deletion is linked with sperm-specific sterility. Multiplex PCR amplicons from sterile worms (Lanes 1-6) and fertile worms (Lanes 7- 10). Al the sterile worms show only 500bp amplicon while the rest amplify both, 700 and 500bp products (except lane-7). 108 translocation cannot survive, a population of heterozygous spe-44(ok1400)/nT1 worms with glowing pharynx and homozygous spe-44(ok1400) worms with non-glowing pharynx is maintained. Segregation of total progeny of spe-44(ok1400)/nT1 and spe- 44(ok1400) dpy-20(e1282)/nT1 balanced lines at 15 0 C and 25 0 C was tested. Proportion of homozygous spe-44(ok1400) or spe-44(ok1400) dpy-20(e1282) to heterozygous over nT1 in the F1 progeny was 1:2 as expected, but no dead embryos for homozygous nT1 were observed in both the strains tested. 3.3.4 Sperm-specific defect In C. elegans, more than 60 mutations have been reported to afect spermatogenesis, which could be broadly categorized into developmental and functional defects. To determine which stage during sperm development is afected by the spe- 44(ok1400) deletion, microscopic analysis was performed. Both wild type and heterozygous hermaphrodites showed normal sperm in their spermathecae (Figure 3-8A). To our surprise, the spermathecae of homozygous worms were without any sperm, as shown in Figure 3-8B. There could be two possible explanations for the lack of sperm in the spermathecae of homozygous spe-44(ok1400) hermaphrodites. One possibility is that the germline in mutant worms never initiate sperm fate as observed for fem-1 loss of function alele hc17 (Nance et al., 1999). The other possibility is that the sperm development is defective in spe-44(ok1400) worms and as a result they get swept out of spermathecae as the hermaphrodites reach adulthood. 109 Figure 3-8: Adult hermaphrodite spermatheca. Wild type (A) and homozygous spe-44(ok1400) (B) spermathecae. Arow indicates mature spermatozoa and arowhead indicates absence of spermatozoa. (100X magnification). 110 The hermaphrodite germline initiates sperm development during the L4 larval stage. To determine if homozygous spe-44(ok1400) worms initiate spermatogenesis or not, hermaphrodites were examined at the L4 larval stage. In heterozygous spe-44/+ hermaphrodites, the germline contains a mixture of spermatocytes and diferentiated spermatids in the most proximal region of the gonad (circle in Figure 3-9A). Spermatocytes also were observed in the homozygous spe-44(ok1400) hermaphrodites, but no spermatids were present. Even after the germline had switched to oocyte diferentiation in these worms, only spermatocyte like cels were observed in the proximal-most part of the gonad Figure 3-9B. These spermatocyte-like cels are refered to as ?terminal spermatocytes? hereafter. Microscopic analysis of homozygous spe- 44(ok1400) adult males also showed similar terminal spermatocytes in the germline with no diferentiated spermatids. 3.3.5 Sperm development is arrested in meiosis The spe-44(ok1400) mutant germline does not form functional spermatids. Cytological analysis of the germline was performed to check the developmental progresion of the germline through spermatogenesis. Young adult hermaphrodite and adult male gonads were disected and observed under DIC microscopy. Wild type gonads showed typical spermatids (arows Figure 3-10, A-B) whereas spe-44 gonads contained cels of spermatocyte size but no spermatids were observed (Figure 3-10, C-D). Some of the spermatocytes from the adult male gonad had a clover-leaf like structure (Figure 3-10D, inset), indicating that some of the cels atempted to divide. 111 Figure 3-9: Proximal region of L4 hermaphrodite gonad. DIC micrograph showing mature spermatids in wild type (A) and spermatocyte-like cels in spe-44 (B) proximal region of the gonad. (100X magnification). 112 Figure 3-10: DIC micrograph of disected gonads. Disected gonad from wild type hermaphrodite (A) and male (B) with spermatids (arow) at the proximal region. The proximal region of the spe-44 disected gonads (C and D) show only spermatocyte like ?terminal spermatocytes? (arowheads) in the proximal region instead of mature spermatids. The inset in panel D shows one of the rare events when terminal spermatocyte atempts to divide. 113 DAPI staining of these disected gonads revealed that the terminal spermatocytes typicaly contained 4 condensed nuclei (Figure 3-11, C-D). The morphology of the condensed nuclei within the cel is very similar to the nuclear morphology of mature wild type spermatids (Figure 3-11B). The presence of 4 condensed nuclei within the cel indicates that the spermatocyte completed karyokinesis during both meiosis I and I. However, the spermatids never separate from the residual body, although some do initiate the atempt (Figure 3-10D, inset). Thus, the spe-44 mutant fails at cytokinesis, and sperm development in both male and hermaphrodite germlines arests as a terminal spermatocyte just prior to the separation of haploid spermatids. Earlier progresion of the germline through pachytene until the primary spermatocyte stage appears normal in spe- 44 worms (Figure 3-12) compared to the wild type germline (Figure 3-2A). The motile pseudopod is necesary for maintaining localization to the spermatheca. As sperm development is not completed in the spe-44 hermaphrodites, crawling spermatozoa are never formed and the terminal spermatocytes get swept away from the spermatheca with the pasing oocytes (Figure 3-13). This phenomenon leads to empty spermathecea as the hermaphrodite reaches adulthood as shown in Figure 3-8B. 114 Figure 3-11: spe-44 terminal spermatocytes with 4 condensed nuclei. Isolated wild type sperm (A) showing single nucleus each (B). Terminal spermatocytes from spe-44 male gonads (C) showing four condensed nuclei within the cel (D). 40X magnification. 115 Figure 3-12: Early meiotic progresion of spe-44 germline. Disected gonad of spe-44 adult male stained with DAPI shows normal ?bowl of spagheti? patern of pachytene nuclei and condensed nuclei of primary spermatocytes. (100X magnfication) 116 Figure 3-13: Spermatocyte swep from the spe-44 young hermaphrodites. Lower panel shows the oocyte (arowhead) sweping terminal spermatocyte cel mas (outlined area) out from the vulval opening (arow). 117 3.3.6 Spermatogenic fate is determined in spe-44(ok1400) germline As a putative transcription regulator of sperm gene expresion, it is posible that spe-44 plays a functional role during diferentiation of the germline or alternatively, in determining the fate of the early germline (L3 stage) to be spermatogenic. If the second explanation is correct, the spermatocyte-like cels observed in the proximal gonad of the L4 hermaphrodites could be cels undergoing meiosis without a specific fate determined. In wild type hermaphrodites, proximal germline cels are already determined to be sperm by the L4 larval stage. One of the first prominent sperm-specific genes to be expresed is the major sperm protein (MSP). The protein can be detected early in primary spermatocytes with anti-MSP monoclonal antibody. MSP is expresed solely during sperm development; for example, MSP is not detected in fem-1 loss-of function mutants where the germline is always oogenic (Nance et al., 1999). Western analysis with anti-MSP antibody on total protein extract from spe-44/nT1 and spe-44 adult males showed expresion in both the worm strains (Figure 3-14). The total signal intensity in the protein extract from spe-44 worms is reduced compared to heterozygous spe-44/nT1 worms. As the germline in spe-44 does not form terminaly diferentiated spermatids, the total number of cels with sperm fate is reduced compared to the heterozygous strain. This could lead to reduced signal in Western analysis. Nevertheles, the fate of the spe-44 germline is already determined to be spermatogenic since MSP is expresed. 118 Figure 3-14: Western analysis of spe-44 males with Anti-MSP. MSP is expresed in both spe-44/nT1 (lane 2) and spe-44 (lane 1) males. In vitro expresed MSP with before and after induction was run as a control (data not shown). 119 3.3.7 spe-44 is expressed in pachytene germline Spermatogenesis is arested as terminal spermatocytes in spe-44 mutant worms. Acording to the hypothesis proposed in the Introduction section, SPE-44 is a putative transcription regulator of sperm gene expresion. The observed arest could be due to reduced or absent expresion of downstream targets of spe-44, or inappropriate expresion if SPE-44 acts as a represor. If this hypothesis is true, spe-44 itself should be expresed earlier in the germline during a narow window of development. spe-44 mRNA expresion was tested by in situ hybridization with a spe-44 cDNA probe on disected gonads. Gonads from L3, L4 and young adult hermaphrodites of fem- 3(q20) (which make only sperm) and fem-1(hc17) (which make only oocytes) worms were fixed and hybridized with the probe. fem-3(q20) gonads from the L3 stage of development show a very strong hybridization signal in the early meiotic germline. The average signal intensity observed in the similar meiotic region in L4 germline is reduced compared to L3 average intensity (Figure 3-15, A-B). The signal is altogether lost in the young adult germline (Figure 3-15C). None of the germlines of fem-1(hc17) worms show any hybridization signal (Figure 3-15,D-F). Thus, the oogenic germline does not expres spe-44 during any developmental stages, consistent with the microaray data from Reinke et al. (2004). Higher magnification images of the germline with DAPI show the hybridization signal overlaps nuclei with the ?bowl of spagheti? patern, which is characteristic of condensed chromosomes within the pachytene stage of prophase I of 120 Figure 3-15: In situ hybridization on disected gonads with spe-44 anti-sense probe. fem-3(q20) gonads in L3 (A), L4 (B) and adult (C) stage with succesively reduced hybridization signal (black arows). fem-1(hc17) L3 (D), L4 (E) and adult (F) gonad with no hybridization signal. 121 Figure 3-16: Spatial pattern of spe-44 expresion in the fem-3 adult germ line with respect to cel cycle stage. The DIC image on the left shows the spe-44 mRNA expresion (arow) and the right panel is a corresponding DAPI image showing nuclear patern. The expresion is exclusively in pachytene stage of meiosis I. 122 meiosis I (Figure 3-16). The signal appears as soon as the germline enters meiosis and is expresed in a very brief window during meiosis, temporaly and spatialy just prior to the specification of spermatogenesis. Although spe-44 is expresed in the pachytene zone, its function is not necesary until later stages of meiosis, as indicated from the morphological defect. 3.3.7.1 Microinjection rescue of spe-44 sperm sterility The wild type spe-44 gene was cloned with approximately 1kb upstream and 700bp downstream sequence. The construct along with the rol-6 marker was injected in heterozygous spe-44(ok1400)dpy-20(e1282)/+ hermaphrodites. Dumpy and rolling worms were picked as transgenic and were screned for fertile hermaphrodites as a rescue of Spe. Two out of seven stably transmited lines partialy rescued the spe- 44(ok1400) linked sterility, with the total viable progeny count of 62 ? 19 and 50 ? 35, respectively. 3.4 Discusion C25G4.4 was identified as a putative transcription regulator based on its homology to known transcription factors, and had been shown to be up-regulated during spermatogenesis in C. elegans (Reinke et al. 2000). The deletion alele of the gene showed the Spe phenotype and hence the gene C25G4.4 was named spe-44. Detailed cytological analysis revealed that sperm development is initiated in spe-44 mutant worms but arests as terminal spermatocytes. The gene is expresed in a very specific spatial and temporal patern imediately prior to sperm production. 123 C25G4.4 was clasified as a putative transcription factor based on its homology with proteins containing the SAND domain. In C25G4.4, the domain encompases amino acids 65-150 and shows 37% identity with the SAND domain in other proteins. Speckled protein 100KDa (Sp100), AIRE-1 (AutoImune Regulatory-1), NucP41/75 and DEAF-1 (Deformed Epidermal Autoregulatory Factor-1) are the founding members that share the SAND domain. Several proteins that contain this 80 amino acid long SAND domain with the conserved KDWK core sequence are asociated with various diseases. AIRE-1 is an autoimune regulator type 1 protein that when mutated causes various autoimune syndromes (Gibson et al., 1998; Park et al., 2003). The speckled protein family encodes components of nuclear bodies, which are linked with neurodegenerative disease and acute promyelocytic leukemia (Bloch et al., 1996; Hodges et al., 1998). One of the proteins of this family, Sp110, has been proposed to function as a nuclear hormone receptor transcriptional co-activator (Bloch et al., 2000). Although the SAND domain does not show any considerable homology to known DNA-binding motifs, its ability to bind to specific DNA sequences and to regulate transcription is wel established. The SAND domain in the proteins DEAF-1 (Gross and McGinnis, 1996), NUDR (Nuclear DEAF-1 Related) (Huggenvik et al., 1998), and GMEB (Glucocorticoid Modulatory Element Binding) (Christensen et al., 1999) have been proposed to mediate DNA binding in a sequence-specific manner. The SAND domain of NUDR has been shown to specificaly bind to TCG repeats (Michelson et. al., 1998 and Bottomley et. al., 2001), the SAND domain of GMEB binds ACGT core sequence (Surdo et al., 2003) and AIRE-1 binds ATGTA or TATA motifs (Kumar et al., 2001). The SAND domain in NUDR and DEAF-1 bind to the TCG 124 signature sequence as a monomer in vitro (Bottomley et. al., 2001). It is interesting to note here that the same domain in diferent proteins shows diferent binding specificities towards the nucleotide sequence. Thre-dimentional structure of the SAND domain has been resolved for Sp100 using NMR (Bottomley et. al., 2001) and for GMEB with X-ray crystalography (Surdo et. al., 2003). It reveals a novel fold for DNA binding (Wojciak and Clubb, 2001). Known DNA-binding domains contain helix-loop-helix or similar motifs, whereas the SAND domain consists of highly conserved positively charged ?-helix with KDWK motif and ?-strands. The motif KDWK is esential for DNA binding and single amino acid mutations in the motif abolish DNA binding in vitro (Bottomley et. al., 2001, Surdo et. al., 2003). Any mutation in the motif alone eliminates NUDR-dependent transcriptional represion in vivo (Bottomley et. al., 2001). Based on the known role of the SAND domain in transcription regulation, it is highly likely that SPE-44 plays a similar role in C. elegans. Expresion of spe-44 is restricted to the germline just prior to the onset of sperm production, suggesting a regulatory role in sperm-specific gene expresion. Northern blot analysis of rat NUDR also shows high expresion in testicular tisue, specificaly in spermatocyte cels (Huggenvik et. al., 1998). Human homolog of NUDR contains a bipartite Nuclear Localization Signal (NLS) as shown in the Figure 3-17, and mutation of the initial residues of the signal sequence eliminates the nuclear localization of hNUDR (Huggenvik et. al., 1998). spe-44 is also predicted to contain a monopartite NLS (based on the prediction program MultiLoc; Nair et al., 2003), which maps to the exact same 125 Figure 3-17: CLUSTALW 2.0.5 multiple sequence alignment. SPE-44 sequence aligned with its homolog hNUDR, which also shows testicular expresion in rat testis. The aqua box is the core sequence of SAND domain. The black arows show the esential amino acids for nuclear localization. 126 location as that of NUDR (Figure 3-17, arows), indicating SPE-44 to be a putative nuclear protein. Further, the study presented in this chapter strengthens the hypothesis that spe-44 is a putative transcription regulator of sperm-specific genes. Hermaphrodites carying homozygous deletion alele of C25G4.4 lay unfertilized oocytes giving a clasic SPE phenotype; hence the gene asignment, spe-44. Cytological analysis of these homozygous worms shows that loss of spe-44 causes arest of gamete development during spermatogenesis. Germline proliferation during spermatogenesis is normal until primary spermatocytes are formed. Arest is observed at the cytokinesis step during meiosis and it specifies the precise window during sperm development where spe- 44 function is esential. Primary spermatocytes initiate meiosis and complete karyokinesis giving four condensed nuclei within the cel (the ?terminal spermatocyte? phenotype). Cytokinesis is either never initiated or not completed in these terminal spermatocytes. In contrast, the oogenic germline is completely unafected by the loss of spe-44. Thus, spe-44 sems to be esential during spermatid formation but not for oogenic development of the germline. spe-44 is expresed in the spermatogenic germline just prior to the time when germline starts diferentiating into sperm. Germline fate to spermiogenesis is determined prior to the expresion of spe-44, as complete loss of spe-44 does not afect MSP expresion, a sperm-specific protein. In situ hybridization shows strong and uniform expresion of spe-44 in the pachytene zone during the L3 larval stage when the spermatogenic germline transits from the transition zone and enters meiosis. The expresion of spe-44 occurs in very restricted spatial context and it is also temporaly transient. L4 germlines show reduced spe-44 expresion and by adulthood, expresion 127 disappeared completely. Rat NUDR also showed a similar patern of expresion that was limited to spermatocyte cels and disappeared in later stages of development (Huggenvik et. al., 1998). Thus, spe-44 is not esential for sperm fate determination or initiation of meiosis but is required for diferentiation of the spermatogenic germline into spermatids. As mentioned earlier, similar arest as terminal spermatocytes is observed in five other Spe mutations; spe-4, spe-5, spe-39, spe-26 and cpb-1. The first thre proteins are required for the FB-MO morphogenesis and their loss of function afects the asymmetric distribution of FB-MO components during completion of meiosis. spe-4 encodes a membrane protein that is a member of the presenilin family implicated in Alzheimer?s disease onset (L'Hernault and Arduengo, 1992). Null aleles of spe-4 lead to terminal spermatocyte phenotype due to the defective asymmetric partitioning of the FB-MO organeles. spe-5 encodes an ortholog of subunit B of the cytoplasmic (V1) domain of vacuolar proton-translocating ATPase and mutations in spe-5 lead to misegregation of tubulin at the end of the meiotic division leading to terminal spermatocyte arest (Machaca et. al. 1997). spe-39 mutants produce terminal spermatocytes that lack MOs; this gene encodes a cytoplasmic protein of unknown homology (Zhu and L'Hernault, 2003). It is also possible that completion of the asymmetric distribution of celular components is esential for initiation of cytokinesis after karyokinesis and the lack thereof results in the arest of cytokinesis. As observed in spe-26, a kelch homolog in C. elegans, null mutation disrupts the segregation of various components like actin and also arests the spermatocytes with multiple condensed nuclei (Varkey et. al., 1995). 128 cpb-1, which encodes a cytoplasmic polyadenylation element binding protein (CPEB), has a more general functional role and is involved in translational control of sperm-specific genes (Luitjens et. al., 2000). The cpb-1 mutation causes development to stal at the primary spermatocyte stage and they never complete the meiotic divisions, in contrast to the previously described Spe mutations. Al of these five mutant terminal spermatocytes look morphologicaly similar (Figure 3-18) to the terminal spermatocytes observed in spe-44. Although their mechanistic action is required at the same spatial and temporal context, there is no correlation in the molecular nature of these gene products and in their modes of action. It is very tempting to speculate that one or more of these genes could be downstream targets that are transcriptionaly regulated through spe-44. If this hypothesis is true, the expresion of these downstream targets should occur spatialy and temporaly after spe- 44 expresion. The microaray data does show that spe-4, spe-5, spe-26 and cpb-1 are up- regulated specificaly during spermatogenesis (Reinke et. al., 2000). The spatial patern of expresion is known for thre of these five targets. spe-39 was shown to be expresed throughout the germline (Zhu et. al., 2003), while spe-26 mRNA is expresed in spermatocytes and the earlier spermatogonial cels as wel (Varkey et. al., 1995). Acording to their spatial context, spe-39 sems to be expresed earlier than spe-44 and is les likely to be a downstream target of spe-44, while spe-26 could be a potential target of spe-44. On the other hand, cpb-1 mRNA is expresed in both the spermatogenic and oogenic germline. During spermatogenesis, it is expresed just distal to the developing spermatids as the imunohistochemistry data suggests (Luitjens et al., 2000). In the 129 Figure 3-18: SPE mutations with terminal spermatocyte phenotype. Wild type primary spermatocyte with single nucleus (left) and with four budding spermatids (right) showing DAPI stained nuclei. Compared to the aberations caused by cpb-1, spe-26, spe4, spe-5 and spe-39 mutations (Luitjens et al., 2000, Varkey et al., 1995, Arduengo et. al., 1998, Machacha et. al., 1997, Zhu et. al., 1997). 130 germline context, this region imediately follows the pachytene zone where spe-44 is expresed. Therefore, it is possible for cpb-1 to be diferentialy regulated by spe-44 with or without co-factors during spermatogenesis. cpb-1 protein controls the translation of the target mRNAs by binding to the poly(A + ) tail of specific mRNAs. The downstream targets of cpb-1 in the spermatogenic germline are not known. Translation of spe-44 itself could very wel be regulated by cpb-1 and thereby lead to the terminal spermatocyte phenotype. It wil be interesting to se if either gene is regulating the other. The spatial expresion patern in the germline for the remaining 2 genes, spe-4 and spe-5 is not yet known. Quantitative RT-PCR experiments are ongoing to determine their expresion levels in the spe-44(1400) background to validate if indeed their expresion is regulated via spe-44. SPE-44 is a potential transcription factor to regulate sperm-gene expresion. In that case, it is likely to be regulating more than these five genes. The limitation of the current work is that not al the targets of SPE-44 are identified. The putative targets of spe-44 could be uncovered by performing microaray expresion analysis on the homozygous spe-44(ok1400) and compare the data set with microaray data of fem- 3(q20) enriched genes. The genes enriched in fem-3 but mising or downregulated (or up- regulated) in spe-44 background would be the strong candidates for spe-44 mediated regulation, either directly or indirectly. This data set of putative targets could be used for promoter analysis to predict the putative binding site for spe-44. The nucleotide sequence present commonly in the promoter region of most or al of the putative targets is likely to be a binding site for SPE-44. This putative binding site could be validated with gel retardation asays. An 131 alternative approach to find the putative binding sites is CHIP-Chip asay. This approach would need a strong antibody to pull down SPE-44 along with its bound DNA from the nuclear extracts of the spermatogenic germline. The putative binding sites of SPE-44 could be validated in vivo using reporter asays and transgenic rescue experiments. As mentioned in the introduction, about 60 of the sperm-specific genes have been studied in some detail. If any of these genes are revealed to be putative targets of spe-44, those could be employed for in vivo validation of results. For example, transgenic rescue of sterility of spe-26 mutants has been demonstrated. The putative promoter of spe-26 could be mutated to se if it can recapitulate the rescue of sterility caused by spe-26 after microinjection. This complete study wil definitely provide insights about transcriptional regulatory aspects of spermatogenesis in C. elegans. 132 Chapter 4 Discusion Celular diferentiation into specialized cel types during development is brought about by temporal and spatial regulation of gene expresion. Understanding the complexities of gene regulation is key to understanding the development of an organism. As many examples of diseases arise due to mis-regulation of gene expresion, studying the underlining mechanisms of gene regulation also helps to uncover the disease principles and can lead towards the cure. In C. elegans, spermatogenesis; the diferentiation of a unicelular sperm from a constitutively proliferating germline, provides a good model system to study cel diferentiation in a developmental context. This disertation presents the study of mutations in two distinct genes isolated from screns for spermatogenesis-defective mutations. Both were initialy believed to represent aleles of putative transcription factors and thereby thought to control sperm diferentiation or a part of the proces. The first alele studied, uba-1(it129), was predicted to be an alele of the transcription factor elt-1 based on the similar phenotypes and map position (refer to Appendices Section A2). elt-1, a GATA transcription factor, was known to be expresed and to play a role during spermatogenesis. Detailed genetic analysis and mapping proved it129 to be an alele of the ubiquitin-activating enzyme (E1) and not a transcription factor. Analysis of uba-1(it129) led to the discovery of a novel role for ubiquitination in sperm development and function. It also revealed the role of ubiquitin conjugation in various developmental proceses of C. elegans as described in Chapter I. 133 Ubiquitination has been implicated in various roles during spermatogenesis in mamals, including humans (Barends et al., 1999). The ubiquitin system is intricately linked with DNA repair, regulation of meiotic chromatin, mitochondrial degradation and sperm quality control (Reviewed in Barends et al., 2000). Mouse and human spermatogonia have been shown to expres spermatogenesis-specific E1 isoforms (Kay GF 1991, Mitchel MJ 1992, Zhu H. et al. 2004). The mouse spermatogenesis-specific E1 isoform Ube1y is encoded on the Y chromosome, and deletion of the Ubel1y leads to spermatogenic failure despite expresion of Ube1x isoform (Odorisio et al., 1996). The proposed reason for an additional E1-encoding gene devoted for sperm development is that it serves to increase UBE1 production at a time of high demand (Odorisio et al., 1996). Although a role for ubiquitination during spermatogenesis is already indicated, the uba-1(it129) alele in C. elegans does not correspond precisely with any of the earlier reported roles for E1. Meiotic progresion through spermatogenesis is normal and morphologicaly normal sperm develop in the mutant worms. These uba-1(it129) mutant sperm are sterile as they are incapable of fertilizing oocytes. Recent evidence from sea urchin experiments indicate a role for the ubiquitin-proteasome pathway for penetration through the viteline layer of the oocyte by the acrosome-reacted spermatozoon (Sawada et al., 2002; Yokota and Sawada, 2007). Drug-induced inhibition of the 26S proteasome inhibited fertilization in these experiments. It is possible that the uba-1(it129) mutation impairs sperm-oocyte interaction because of reduced ubiquitination of surface proteins involved in mediating the interaction. It wil be interesting to investigate the profile of ubquitin-tagged protein conjugates in wild type and uba-1(it129) sperm. A diferential 134 profile could lead us to the proteins involved in sperm-oocyte interactions and help us to understand the ever-complex fertilization phenomenon. The uba-1(it129) mutation afects sperm function during the sperm-oocyte interaction but does not lead to any meiotic aberations during sperm development as observed for mutations in ubiquitin-conjugation system in other model organisms. One other aspect of C. elegans development where ubiquitin conjugation has been implicated to be esential is meiotic progresion after fertilization and further embryo development. Many diferent mutations in the anaphase-promoting complex (APC), a multi-subunit E3 ligase complex led to this discovery. Severe mutations of the APC components lead to metaphase arest (Mat phenotype) during the first meiotic division in the fertilized oocyte (refer to Chapter I). As the ubiquitin-activating enzyme (E1) works upstream of the APC complex, one would expect that impairment of E1 function would lead to a similar phenotype. Instead, uba-1(it129) was shown to slow down the meiotic progresion rather than aresting it as the Mat phenotype (chapter I, Figure 7). This evidence leads to the conclusion that uba-1(it129) is a hypomorph of E1. Some of the other phenotypes manifested by the same alele show drastic aberations in distinct developmental pathways like larval lethality and male paralysis. Why does a single point mutation in E1 enzyme lead to such diversity in the severity of the defects? The vast number of E2 and E3s functioning downstream of E1 increase the complexity of the ubiquitin conjugation system. Distinct E3 complexes have been implicated in separate developmental pathways. The interaction betwen distinct E2-E3 complexes and E1 enzyme could difer in the their respective afinities. As a result, it is possible that diferent subsets of E2-E3 complexes bind diferently to the E1 mutation. 135 Similar mechanisms are implicated for mutualy excusive cel cycle arest phenotypes manifested by distinct point mutations of E1 in human cel lines (Ayusawa et al., 1992; Jentsch et al., 1991; Zacksenhaus et al., 1990) and antagonistic efects on cel growth caused by distinct mutations in Drosophila E1 (Le et al., 2008). Recent progres in our understanding of ubiquitin conjugation has revealed increasing complexity of the system as E1-like enzymes known to activate Ubls have been shown to activate ubiquitin as wel (Chiu et al., 2007). There are four known ubiquitin-activating enzyme-like proteins predicted in the C.elegans genome, but with the emergence of novel ubiquitin-like peptides and their respective E1-like enzymes, it is not unlikely to find more E1-like enzymes encoded in the genome. If similar cross-activity of E1-like enzymes as reported in other organisms exists in C. elegans as wel, impairment of E1 enzyme itself would not afect the entire ubiquitin-conjugation as severely as one would speculate. Concentration of fre ubiquitin in the cel is critical for cel survival and proper function of the proteasome. How the concentration of fre ubiquitin is maintained at appropriate levels (i.e., how ubiquitin homeostasis is maintained) is stil not understood. Transcriptional and translational control of the polyubiquitin gene, the rate of fre ubiquitin activation, and the rate of deubiquitination from the target proteins al coordinately contribute towards ubiquitin homeostasis. For example, the levels of celular ubiquitin regulate the celular abundance of proteasome-asociated deubiquitinating enzyme Ubp6; under the conditions of ubiquitin depletion, Ubp6 levels increase (Hanna et al., 2003). In the absence of Ubp6, the half-life of ubiquitin is dramaticaly reduced and the cels become deficient in fre ubiquitin levels (Chernova et al., 2003; Hanna et al., 136 2003; Legget et al., 2002). Ubiquitin-activating enzyme also regulates the fre ubiquitin by controlling the rate of activation. In the mamalian cel line containing ts85 mutation in E1, de novo synthesis of ubiquitin is reduced indicating some fedback mechanism for ubiquitin expresion (Finley et al., 1984). Any deviation from homeostasis is realized as a stres by the cel, which then responds to this stres by altering proteasome subunit composition (Hanna and Finley, 2007). Ubiquitin-dependent upregulation of Ubp6 results in greater loading of proteasomes with Ubp6, presumably resulting in greater eficiency of ubiquitin recycling at the proteasome (Hanna et al., 2003). Opposite to this scenario of ubiquitin depletion, functional impairment of ubiquitin-activating enzyme would lead to increase in the fre ubiquitin pool, which also could lead to alteration in the subunit composition of the proteasome. The altered composition of proteasome might change its selectivity for particular E3 complexes and their target proteins, which could lead to inhibition of degradation in a selective manner. Thus, it is posible that the reduced activity of E1 enzyme in the uba-1(it129) mutant worms alters ubiquitin homeostasis. Some of the defects observed during development of uba-1(it129) mutant worms might be manifested as an indirect efect of the E1 mutation. The diferential severity of the phenotypes could reflect the extent of deviation in the proteasomal subunit composition. It wil be interesting to monitor the fre ubiquitin pool at diferent developmental stages in wild type and mutant worms and also compare the 26S proteasome composition at those respective stages. The second mutant studied is a deletion alele of a putative transcription factor with a SAND domain, spe-44. It is required for sperm diferentiation, as the deletion 137 arests spermatogenesis during cytokinesis after completion of meiotic karyokinesis. This terminal spermatocyte phenotype manifested by the spe-44 deletion has also been reported for five diferent spe genes as discused in Chapter II. None of these five genes encode a transcription factor. spe-44 and these genes could be part of the same regulatory cascade as depletion of any of the genes leads to the arest at the same developmental point during sperm diferentiation. The expresion of one or more of these genes could very wel be regulated by spe-44, along with additional putative targets for spe-44. Microaray expresion analysis in the spe-44 deletion background would reveal the network of players involved in post-meiotic diferentiation of C. elegans sperm. How spe-44 itself is regulated is an important question. The expresion analysis showed transient expresion of spe-44 during the spermatogenic phase of the germline in a very narow spatial window (Figure 3-14 and 3-15). To achieve this transient spatio- temporal expresion, spe-44 has to be under tight regulatory control. microRNAs (miRNAs) could be regulating the depletion of spe-44 mRNA observed in the late meiotic spermatogenic germline. miRNAs are single-stranded RNA molecules with approximately 21 or 22 nucleotides. They encode sequences complementary to the sites in the 3' untranslated region (UTR) of their target mRNAs. They function as inhibitory regulators of mRNAs either by decreasing target mesenger RNA levels or by directly inhibiting their translation (Reviewed by Boyd, 2008). miRNAs are also proposed to destabilize the target mRNA via deadenylation of the poly- A tail (Standart and Jackson, 2007; Wu L et. al., 2006). Registry of annotated miRNA targets predicts thre miRNAs (cel-miR-34, cel-miR-251 and cel-miR-272) with complimentary sequence in the spe-44 5? UTR (http:/microrna.sanger.ac.uk/cgi- 138 bin/targets/v5/detail_view.pl?transcript_id=C25G4.4). The rapid depletion of spe-44 mRNA observed in the germline at pachytene exit could be brought about by one or more of these miRNAs, although further investigation is necesary to validate these predictions. Spermatogenic fate in the male and hermaphrodite germline is determined by fog- 3, a member of Tob family of transcription cofactors, in conjunction with fog-1, which is a homolog of the cytoplasmic polyadenylation element binding protein (Chen P et al., 2000, Luitjens et al., 2000, Jin et al., 2001). Sperm fate determination is under direct control of the sex-determination pathway transcription factor TRA-1, as both fog-1 and fog-3 promoters contain multiple TRA-1 binding sites (Chen and Elis, 2000; Jin et al., 2001) and fog-3 expresion has been experimentaly shown to be under direct control of TRA-1 (Chen P et al., 2000). FOG-1 protein is expresed from early L3 through the mid-L4 larval stage and disappears from spermatogenic precursors prior to expresion of an early sperm- diferentiation marker, SP56 (Lamont and Kimble, 2007). The extent and duration of fog- 1 directly determines the total number of sperm in the hermaphrodite germline (Lamont and Kimble, 2007). FOG-1 encodes a CPEB homolog, which binds to regulatory elements in the 3? untranslated region of target mRNAs and can either activate or repres their translation as demonstrated for Xenopus homolog (Richter, 2000). It could function as a sperm specification factor by initiating the expresion of transcription factors such as spe-44 and elt-1, which are esential for further diferentiation of the sperm. It wil be interesting to search for CPEB binding regulatory elements (CPEs) in spe-44 UTRs. 139 Along with the expresion initiation, suppresion of the expresion of spe-44 after mid-L4 stage and post-pachytene germline has to be regulated. Acording the mRNA expresion analysis, spe-44 expresion ceases during mid-L4 stage, when the germline switches the fate from spermatogenesis to oogenesis. During the fate switch, sperm-fate- determining factors like fog-1 and fog-3 are also downregulated. If spe-44 is under direct or indirect control of these factors, one would expect that spe-44 expresion also wil go down at the same time. Another possibility is that, as the germline switches the fate to oogenesis, transcription regulators needed for further specification and diferentiation wil be expresed in the meiotic germline. Analogous to antagonistic actions betwen the sub-networks of transcription factors in C-blastomere lineage specifications (Refer to Chapter I; section X), these oogenic transcription regulators could continuously shut down the expresion of spe-44 and other sperm-specific regulators. A few details of the two ends of the regulatory cascade from determination of sperm fate til diferentiation of functional spermatozoa have been characterized. As explained earlier, fog-3 and fog-1 are known factors required to specify the sperm fate. Many diferent genes have ben identified at the end of the cascade, which play a role in sperm activation and functional aspects of spermatozoa (as reviewed in L?Hernault, 2005). The link betwen these two ends of the pathway is stil mising. The transcription factors revealed from the microaray experiments (Reinke et al., 2004) are potential factors to initiate the expresion of the downstream efectors and themselves could be under fog-3 and fog-1 control, either directly or indirectly. The experiments proposed above to understand the regulation of spe-44 expresion and the downstream targets of SPE-44 could provide insights for this mising link. 140 Chapter 5 Apendices 5.1 Western analysis of uba-1(it129) worms to detect ubiquitin- activating enzyme expresion and ubiquitination of proteins. 5.1.1 Introduction uba-1 encodes ubiquitin-activating enzyme (E1) in C. elegans (refer to Chapter I). It is the first enzyme in ubiquitin-conjugation pathway, which leads to poly- ubiquitination of target proteins. The alele it129 ts encodes a point mutation in uba-1. The mutant protein is functional at permisive temperature, but at the restrictive temperature the mutation renders various developmental defects in C. elegans. To detect if the E1 protein levels are altered at the restrictive temperature, I performed Western analysis using anti-E1 antibody. Wild type uba-1 cDNA was cloned in protein expresion vector pET28a and in vitro protein induction was atempted to purify E1 protein to use as a positive control in the Western analysis. Various developmental defects observed in it129 ts mutant worms like embryonic lethality, sperm-specific sterility, male tail aberations are more likely to be afected through independent E3 ubiquitin-ligases (please refer to chapter I discussion section). Since the E1 function is esential at the apex of the ubiquitin-conjugation function i.e. prior to al the E3 action, various defects in it129 ts alele could be due to overal reduction in ubiquitin-conjugation. To test this hypothesis, I performed Western analysis with anti- ubiquitin antibody on total protein extracts from wild type and uba-1(it129 ts )worms. 141 Ubiquitination levels in sperm protein extracts were also analyzed by anti-ubiquitin Western as it129 ts mutation afects sperm function. SP56 antibody was generated in Dr. Samuel Ward?s lab against total sperm isolated from wild type C. elegans males (Ward et al., 1986). It was shown to recognize multiple bands up to 8 from total sperm protein extracts. Our speculation is that this antibody could be recognizing ubiquitin epitope from the sperm proteins. The staining patern of this SP56 antibody was compared with anti-ubiquitin staining patern for total sperm protein. The hybridoma culture supernatant of the SP56 antibody was a generous gift of Dr. Steven L'Hernault. The methods and results for al these Western experiments are discussed in the following sections. 5.1.2 uba-1 cDNA cloning and in vitro protein induction. 5.1.2.1 Methods uba-1 cDNA was PCR amplified from cDNA pool extracted from fem-1(hc17) and fem-3(q20) worms. 3342 bp fragment was amplified using sense primer with NheI site CTAGCTAGCATGACTACATCTGAG and anti-sense primer with BamHI site CGCGATCTAGAAGAGTAGCG. The fragment was cloned into pET28a betwen NheI and BamHI. Selected clones were sequenced to confirm the correct coding sequence. The plasmid clone was transformed in E. coli; strain BL21(DE3). The expresed protein is predicted to be 1113 amino acids from the uba-1 cDNA plus 19 amino acids from vector including 6 histidines. The approximate size of the expresed protein is 126 KDa. 142 One colony of pET28a:uba-1cDNA was grown in LB-kanamycin overnight culture at 37 0 C. Culture was diluted to OD 60nm =0.05 and split in two batches of triplicates of 5 ml. One batch was grown at 37 0 C and second was at room temperature. Both the batches were induced with 0.5 mM, 1 mM and 2 mM IPTG at OD 600nm =0.5. Samples were collected as 300 ?l before induction (T 0 ) and at 1 hour after induction (T 1 ) and then 100 ?l at every 3 hours (T 2 to T 6 ). Final sample of 100 ?l was collected after overnight induction. Collected samples were peleted at 5000 g and the pelet was boiled with 50 ?l 1X sample buffer (100 mM Tris-HCl, pH 6.8, 2% Sodium Dodecyl Sulfate (SDS), 20% Glycerol, 0.2% Bromophenol Blue, 2?10% ?-mercaptoethanol). 20 ?l of each sample was loaded on 4-20% SDS-PAGE gel along with NEB broad-range protein marker (P7702; 2- 212 KDa). 5.1.2.2 Results Wild type uba-1 was expresed from pET28a bacterial vector (Novagen) for in vitro purification. Induction of a protein of molecular weight of 126 KDa was observed within 1 hour when induced with 2 mM IPTG and grown at 37 0 C and in al the overnight grown cultures at room temperature with 0.5 mM, 1 mM and 2 mM IPTG. Protein band at 126 KDa was absent in uninduced (T 0 ) samples. Refer to Figure 5-1. 143 Figure 5-1: Coomassie gel with crude protein extract from bacterial pelet expresing Ce-UBA-1. Lanes 1-4, bacterial protein extracts induced with 2 mM IPTG at 0 hr (lane 1), 1 hr (lane 2), 6 hr (lane 3) and overnight induction point. Lanes 3-7, bacterial extracts after overnight induction with 0.5 mM, 1 mM and 2 mM IPTG. Lane M is the protein marker indicated with the size standards on the right. The arow indicates the induced E1 protein at approximately 126 KDa size. 144 Although the E1 protein band of expected size was observed after IPTG induction, the expresion level neds to be improved to be able to purify E1. Trials are ongoing currently to improve the expresion using diferent growth media. Since the pure protein was not obtained, it was not included in the following Western analysis. Instead, total cel extract from a human cancer cel line was included as a positive control. 5.1.3 Western blot analysis with anti-E1 (ubiquitin-activating enzyme) antibody. 5.1.3.1 Methods 5.1.3.1.1 Total protein preparation from adult worms Synchronized populations of worm strains were raised at appropriate temperature (15 0 C or 25 0 C) in 25cm peptone plates with NA22 bacteria. Worms were washed with M9 and peleted in a tight pelet of 100?l in homogenization buffer (15 mM Hepes pH7.5, 10 mM KCl, 1.5 mM gCl 2 , 0.1 mM EDTA, 0.5 mM EGTA, 44 mM Sucrose) with 1?l of 1M DT and 3?l of protease inhibitor mix (1 ?g Pepstatin, 1 ?g Leupeptin, 4 ?g Aproteinin and 10 ?M G132-26S proteasome inhibitor). The pelet was frozen at - 80 0 . The pelet was homogenized before it was completely thawed using handheld homogenizer with additional 1 ?l of 1M DT and 3 ?l of protease inhibitor mix for about 40 seconds until it was thick slurry. The slurry was boiled for 10 minutes with equal volume of 2X sample buffer (4%SDS, 100 mM Tris-Cl pH 6.8, 20% glycerol). Then it was pased through 26G syringe for 2-3 times and centrifuged at 10,000 g for 10 minutes. 145 The supernatant was stored at -80 0 C. Samples were loaded with 1 ?l of 1M DT and 1 ?l of 10X Bromo-phenol blue solution. 5.1.3.1.2 Total protein preparation from isolated sperm This protocol was adopted from the one developed in Dr. Ward?s lab (Klas, M.R et al. 1981). Large synchronized populations of worms enriched for males (Him strain) were grown til young adulthood at appropriate temperature. Worms were rinsed with M9 buffer twice and left in 5 ml volume of M9 buffer. These worms were pased through sterilized 35 ?m esh screns into 15 cm containing M9 for about 30 minutes. The males being smaler in diameter pas through the mesh. The flowthrough with males was concentrated by centrifugation at 2000 rpm with 2 minutes spin. The males were grown for additional 24 hours on bacteria to maximize sperm production. Purified males were rinsed thre times in PSM (10 mg/ml polyvinylpyrrolidone, 45 mM choline chloride, 25 mM KCl, 10 mM HEPES (K), pH 7.3, 0.1% glucose, 1 mM MgSO 4 , 1 ?g/?l leupeptin, 1 ?g/?l pepstatin A, 5 ?g/?l aprotinin). A plain, sterile microscope slide was placed into 10 cm glas petri dish (sterile) and up to 500 ?l male suspension was dispensed onto the slide. The slurry was chopped with sterile razor blade for five minutes, using slicing motion. The slide and the razor were rinsed with 5 ml of PSM into 15 ml centrifuge tube. The volume was dispensed onto the surface of sterile 10?m scren and rinsed twice with 5 to 10 ml PSM. 10 ml aliquots of the flow through were carefully underlaid with 3 ml 10% (v/v) Percoll in PSM in 15 ml centrifuge tubes with Pasteur pipete. The interface was gently mixed by stiring the pipete tip. The tubes were centrifuged at 500 g for 10 minutes and supernatant was aspirated gently. The 146 sperm pelet was resuspended in 2 ml PSM and was washed twice at 750 g for 3 minutes. Total sperm present in the solution were counted using hematocytometer. The pelet was either frozen at -80 0 C or resuspended in 1X SDS sample buffer to give 5 ?g/?l total protein concentration (3 x 10 7 sperm ? 480 ?g of protein). 5.1.3.1.3 Total protein isolation from human cell line ChaGo-K-1 (ATCC# HTB-168) Total protein from human lung cancer cel line was isolated to use as positive control for the Anti-E1 Western. One flask of confluent culture was a generous gift from Dr. Turko, it washed with 2ml of 1X PBS containing protease inhibitor mix. 1 ml volume was sonicated 3 times at 40% with smalest tip using Branson Sonifier-450. The sample was centrifuged at 120K g for 3 minutes at 4 0 C. The supernatant and the pelet were frozen at -80 0 C. 150 ?l supernatant was used to precipitate protein using chloroform/ethanol precipitation method. Protein concentration was 0.57 ?g/?l as estimated by Bradford method. 5.1.3.1.4 Western Transfer After running the SDS-PAGE gel for protein separation, the gel was equilibrated with 1X transfer buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) with 20% methanol. PVDF membrane was activated in 100% methanol, washed with water and was equilibrated with 1X transfer buffer for 20 minutes. The gel and the PVDF membrane were sandwiched betwen 2 sheets of 3MM Whatman papers soaked in 1X transfer buffer. The entire asembly was sandwiched in a casete betwen 2 sponges soaked in 147 1X transfer buffer and the casete was imersed in the Hoefer tank containing 1.5 liters of 1X transfer buffer keeping the PVDF membrane towards the anode side. The transfer was caried out at 100V for 70 minutes. After transfer, the membrane was blocked in 5% milk (nonfat powdered milk) in TBST (20 mM Tris pH 8, 150 mM NaCl, 0.05% Twen- 20) for 1hr at room temperature. The membrane was washed in TBST for 3 times at 15 minutes interval. It was then incubated overnight at 4 0 C with mouse monoclonal Anti-E1 (Sigma #E3152) at 1:500 dilution in 5% milk-TBST. The next morning, the membrane was washed and then incubated with HRP conjugated Anti-mouse IgG 1 (Stresgen # SAB-100) 1:2000 diluted in 5% milk-TBST for 1 hr at room temperature. The membrane was washed 3 times with TBST and then developed using PIERCE Supersignal substrate (# 34077). It was then exposed to X-ray film in the dark for 1 to 40 minutes. The film was developed using AFP imaging system. 5.1.3.2 Results 5.1.3.2.1 uba-1 protein expression in wild type and it129 ts worms. To determine if expresion level of E1 was afected by the it129 ts mutation, Western analysis was performed on total protein extracts of wild type and mutant males raised at 25 0 C. Human ubiquitin-activating enzyme (E1) was detected at appropriate 117KDa position on the blot (Figure 5-2). Although the predicted size of C. elegans ubiquitin-activating enzyme (E1) is 124 KDa, multiple bands were observed in worm protein samples at 100 KDa, 150 KDa and betwen 150 KDa and 250 KDa. The band sizes observed above 120 KDa could be isoforms of worm E1 as shown for E1 from mamalian system (Cook and Chock, 1992). The Expresed 148 Figure 5-2: Western analysis of total protein from wild type and uba-1(it129 ts ) worms with Anti-E1. 10% SDS-PAGE gel was run with the following protein extracts. Lane 1) 0.6 ?l Cha-Go cel extract, 2) 10 ?g of extract from fem-1(hc17) adult hermaphrodites (produce only oocytes), 3) 10 ?g of extract from fem-3(q20) adult hermaphrodites (produce only sperm), 4) 10 ?g of extract from him-5(e1490) and uba-1(it129 ts )him-5(e1490) adult males grown at 25 0 C and M) the protein marker Biorad # 161-0373 (15-250 KDa) as indicated on the right side. 149 Sequence Tag (EST) database for C. elegans gene annotations also predicts six diferent transcripts from the uba-1 gene. The protein extracts from adult male worms show a diference in the band size at around 150 KDa and 250 KDa compared to adult hermaphrodite protein extracts indicating diferential transcripts or post-translational modifications of E1 in males and hermaphrodites. Confirmatory experiments like MS-MS analysis need to be to done to conclude the identity the protein bands recognized by anti-E1 antibody to be E1. If the protein bands recognized are indeed of E1, the protein expresion level is elevated in it129 ts males compared to wild type males, whereas mutant hermaphrodites show equivalent expresion of UBA-1. This could possibly be the cause for sever sex-specific phenotypes manifested in uba-1(it129 ts ) males. 5.1.3.2.2 uba-1 protein expression in wild type sperm. uba-1(it129 ts ) worms produced morphologicaly normal but functionaly impaired sperm (refer to chapter I). To detect if E1 is expresed in sperm tisue as wel, Anti-E1 Western was performed on the total protein extracted from isolated sperm. The sperm protein extract, even at 30 ?g concentration, did not show any distinct E1 band at expected size (Figure 5-3). There is a faint band in betwen size standard 150KDa and 250KDa in 6 ?l sample lane. Either E1 is not expresed in sperm cels or not to the level enough to detect within the limits of Western analysis. The extraneous bands detected at 37 KDa and 70 KDa could be non-specific or degradation products of E1. 150 Figure 5-3: Western analysis of total protein from isolated sperm for E1. 10% SDS-PAGE gel was run with the following samples. Lanes 1-4, Western blot with anti-E1 antibody (1:500 dilution) exposed for 45 minutes. Lane 1-3) 5 ?g; 20 ?g; 30 ?g of sperm protein sample isolated from him-5(e1490) adult males grown at 25 0 C. Lane 4) 0.6 ?l Cha-Go cel protein extract. Lane M) The protein marker Biorad # 161-0373 (10- 250KDa). Lanes 5) Coomasie stained gel with 20 ?g of total sperm protein from him- 5(e1490) adult males. 151 Spermatids are formed during spermatogenesis by asymmetric distribution of the components betwen four haploid spermatids and a residual body. The components non- esential for spermiogeneis and sperm function are packaged in the residual body. It is possible that the proteins are loaded in the budding spermatids as ubiquitinated form, so that E1 does not need to be packaged in the spermatids. In such scenario, we would not detect any E1 in the Western analysis of total sperm protein. 5.1.4 Western analysis to detect ubiquitination levels in wild type and it129 ts mutant worms. 5.1.4.1 Methods Al the procedures were caried out as described in the section 5.1.3.1. Blocked PVDF membrane was incubated overnight at 4 0 C with mouse monoclonal Anti-Ubiquitin (Stresgen # SPA-203) at 1:500 dilution in 5% milk-TBST. The secondary antibody used was HRP conjugated Anti-mouse IgG 1 (Stresgen # SAB-100) at 1:2000 dilution in 5% milk-TBST. 5.1.4.2 Results 5.1.4.2.1 Anti-ubiquitin Western on serial protein concentrations to determine the sensitivity of the antibody. Serial concentrations of protein extracts from diferent tisues were tested with anti-ubiquitin antibody. Ubiquitin-conjugation as polyubiquitin or monoubiquitin tag is an esential post-translational modification of proteins. The list of proteins regulated by this modification is ever increasing. Multiple protein bands were detected from both the 152 sperm and fem-3 adult worm samples indicating multiple ubiquitinated proteins as expected (Figure 5-4). Ubiquitin is expresed as a precursor polyubiquitin peptide from the polyubiquitin. The C. elegans genome has one polyubiquitin locus, ubq-1 (Graham et al., 1989). It encodes 11 tandem repeats of ubiquitin as an 838 amino acid peptide (Gonczy et al., 2000; Piano et al., 2000). Protein band detected at around 75 KDa could be an unprocesed protein product from the poly-ubiquitin gene. 5.1.4.2.2 Comparative analysis of ubiquitination levels between wild type and it129 ts worms. This Western analysis was performed to compare the status of ubiquitination of proteins in uba-1(it129 ts ) mutant, which is predicted to encode a functionaly impaired ubiquitin-activating enzyme, with wild type worms. Multiple proteins were recognized as ubiquitinated on the blot in al of the protein samples (Figure 5-5). The sperm protein and adult mixed population for hermaphrodites and males protein extract from uba-1(it129 ts ) genetic background do show reduced signal compared to wild type protein extract. This could be a direct result of reduced ubiquitination. The ubiquitination signal in adult uba- 1(it129 ts ) males is either not reduced dramaticaly or the blot is overexposed to make any statement. Although there are few diferences in the band patern observed compared to him-5(e1490) males. 153 Figure 5-4: Standardization of Western analysis with anti-ubiquitin antibody. 10% SDS-PAGE gel was run with the following samples. 5 ?l; 10 ?l; 20 ?l Cha-Go cel protein extract, 30 ?g; 20 ?g; 6 ?g of protein extract from him-5(e1490) male sperm, 20 ?g; 10 ?g; 5 ?g of protein extract from fem-3(q20) adult hermaphrodites producing only sperm and the protein marker (Lane-M) Biorad # 161-0373 (10-250KDa). 154 Figure 5-5: Western analysis with anti-ubiquitin antibody on protein extracts from adult worms and isolated sperm. 10% SDS-PAGE gel was run with 10 ?l Cha-Go cel protein extract (lane-8), 15 ?g of him-5(e1490) sperm protein (lane 2) and uba-1(it129 ts )him-5(e1490) sperm protein (lane 3), 5 ?l of total protein isolated from him-5(e1490) (lane 4) and uba-1(it129 ts )him- 5(e1490) adult males (lane 5) grown at 25 0 C, 5 ?l of total protein isolated from N2 (lane 6) and uba-1(it129 ts ) (lane 7) adult populations containing hermaphrodites and males both and the protein marker Biorad # 161-0373 (lane M). The blot was exposed for 2 minutes. 155 5.1.5 Western blot analysis with SP56 antibody 5.1.5.1 Methods 10% SDS-PAGE gel was run with the following samples at 25mA for 90 minutes. Triplicates of 6?l sperm protein sample (conc. 5 ?g/?l) isolated from him-5(e1490) and the protein marker Biorad # 161-0373 (10-250KDa). Western analysis was performed as described in section 5.1.3.1 with 1:10, 1:100 and 1:200 dilutions of SP56 antibody in 5% milk-TBST. 5.1.5.2 Results Monoclonal antibody SP56, recognizes 8 sperm-specific epitopes (Figure 5-6). It is very unusual for any anti-body to recognize more than one epitope, but SP56 has been shown to recognize a post-translational modification of the subset of sperm proteins (Ward et al. 1986). We speculate this post-translational modification to be poly- or mono- ubiquitination of sperm proteins. But the signal patern observed here for SP56 does not overlap with the patern observed in anti-ubiquitin Western analysis of the same sperm protein extract (Compare Figure 5-6 with Figure 5-5, lane 2). Based on the band patern, it is les likely that SP56 recognizes the ubiquitin epitope. 156 Figure 5-6: Western analysis with SP56 antibody on sperm protein extract. 25 ?g of total sperm protein from him-5(e1490) adult males blotted with 1:200 (lane 1) and 1:100 (lane 2) dilution of SP56 antibody. 157 5.2 Genetic interaction betwen elt-1(zu180) and uba-1(it129 ts ) 5.2.1 Introduction In C. elegans, elt-1 encodes a GATA transcription factor. It has been shown to play an esential role during embryogenesis to specify hypodermal fate and also for the maintenance of hypodermal seam cels in later life (Smith et al., 2005). The alele zu180, whose molecular nature is not known, leads to embryonic lethality when homozygous. Various novel phenotypes were observed in worms treated with elt-1 RNAi apart from the embryonic lethality (Smith J.A. et. al. 2005). L1/L2 larvae hatched from RNAi treated embryos die as lumpy-dumpy larvae. Treated P0 hermaphrodites burst their vulva in adulthood. Overexpresion of elt-1 in transgenic animals makes hermaphrodites hypermotile and eventualy leads to paralysis. Al these phenotypes overlap with the range of phenotypes observed with it129 ts (refer to chapter I). Also, based on elt-1 RNAi on wild type males as described in section 5.2.2, treated males showed similar tail aberations as sen in homozygous it129 ts males. On the genetic map of C. elegans, both the aleles it129 ts and elt-1(zu180) are placed very close to each other on chromosome IV. Prior to the identification of it129 ts as an alele of uba-1, this data led to the speculation that it129 ts could be a temperature-sensitive alele of elt-1 and phenocopies the stage-specific RNAi phenotypes of elt-1. I performed non-complementation analysis to test the alelism of it129 ts with elt-1 using the zu180 alele. The elt-1 mutation did complement al of the phenotypes of it129 ts , indicating that alele it129 ts is encoded in a diferent gene than elt-1. During the course of this study, genetic interaction betwen these two aleles was observed. The next 158 few sections wil discuss the nature of the genetic interaction, novel observations made for zu180 alele and elt-1 mRNA expresion in the germline. 5.2.2 elt-1 RNAi on early larval males leads to developmental defects in their tail organs. 5.2.2.1 Methods RNAi construct (pHS482) was generated by cloning an elt-1 cDNA fragment into pPD129.36 betwen inverted T7 promoters using BamHI and XhoI sites. The cDNA was amplified from fem-3 cDNA pool using primers HES-209 (AAGATCAGAACATGACTACG) and HES-210 (AAGTCGACGATGTTGAATGAG). The clone was then transformed in E. coli strain HT115. Synchronized populations of N2 (wild type) hermaphrodites and males were grown until L2-L3. Sets of 20 hermaphrodites or males were shifted to NGM plates expresing elt-1 RNAi construct or control HT115 strain in triplicates. The worms were maintained on RNAi plates until late adulthood and the worms were examined for the phenotypes. 5.2.2.2 Results elt-1 RNAi on hermaphrodites reproduced al the previously reported phenotypes like F1 embryonic lethality, lumpy-dumpy F1 larvae and vulval bursting in P0 (treated) hermaphrodites (Smith et al., 2005). None of these phenotypes were observed in the hermaphrodites treated with control RNAi, confirming the specificity of elt-1 RNAi. 159 elt-1 RNAi was performed on males to check for possible novel phenotypes. The males treated with elt-1 RNAi did show aberations in the tail development. The tail tip, cuticular fan and the sensory rays are reduced in size compared to wild type male tail at the equivalent life stage as shown in Figure 5-7. The posterior body portion in elt-1 RNAi treated males show an increased number of vacuoles compared to control-treated wild type males. Thus, loss of elt-1 in males afects the same organs of the tail as in it129 ts , although the severity of the defect is diferent. 160 Figure 5-7: Comparison of male tail structures betwen wild type and elt-1 RNAi treated wild type males. The tail of elt-1 RNAi treated male shows reduced cuticular fan and no sensory rays as compared to the wild type tail. 161 5.2.3 elt-1 (zu180) homozygous strain behaves differently at 15 0 C and 25 0 C. The alele zu180 is maintained as balanced lines in strains J398 (unc-24(e138) daf-14(m77)/elt-1(zu180) dpy-20(e1282) IV) and J1129 (elt-1(zu180) unc-43(e408)/unc- 24(e138) dpy-20(e1282) IV). Both the strains produce reduced proportion of homozygous elt-1 progeny. The reduction in the proportion is severe at 25 0 C than at 15 0 C (Table 5-1). At 25 0 C, though, both the strains only lay 6-13 dead embryos per 147 average total progeny (4-9%). Proportion of the surviving F1 progeny is not altered in case of both the strains. This indicates that the higher temperature has some efect on the quality of the oocytes and/or sperm carying elt-1(zu180) alele, making the homozygous elt-1 embryos disappear from the pool of the F1 progeny. 162 Strain J1129: elt-1unc-43/unc-24daf-14 Observed selfed progeny Expected 15 0 C 25 0 C elt-1unc-43 (dead eggs) 25% 28?1.2 (14%) 6?4.2 (4%) Parental (wild type) 50% 131?24.6 (65%) 88?21.7 (62%) unc-24daf-14 25% 43?5.1 (21%) 39?9.6 (27%) Total progeny 202?14.2 143?22.5 Table 5-1: Proportion of genotypes in the F1 progeny from the strain carrying elt- 1(zu180). Strains carying elt-1(zu180) alele show anomalous bevavior at 25 0 C. The heterozygous strains throw only 4% dead embryos insead of 25% (expected) of total progeny, representing homozygous elt-1(zu180) worms. 163 5.2.4 in situ hybridization to check elt-1 mRNA expression pattern in sperm producing germline. 5.2.4.1 Methods N2 hermaphrodites and males, fem-3(q20) and fem-1(hc17) worm populations were bleached and hatched on NGM plates overnight at 15 0 C. The hatched L1 larvae were shifted to NGM plates with food at 25 0 C until the appropriate stage. fem-3(q20) hermaphrodites produce only sperm while fem-1(hc17) produce only oocytes at 25 0 C 5.2.4.1.1 Worm dissections Synchronized populations of worm strains were obtained at L4 or adult stage. The worms were collected in PBS with 0.25 mM leavamisole. About 200-300 worms were disected with a 20-gauge needle to extrude the gonad arms and then collected in sterile 3ml glas culture tube with the conical bottom. 5.2.4.1.2 ssDNA probe synthesis elt-1 cDNA was amplified (1.3 kb fragment) from the clone pHS482 using anti- sense primer HES-395 (AAAGCTGTCGACGATGTCTGAATGAGAG) and sense primer HES-398 (AAAGCTCTCGAGCAGAACATGACTACG). Using this cDNA as a template, single stand was linearly amplified using either 3? anti- sense or 5? sense primer with a DIG-labeled mix acording to the instructions given in PCR DIG probe synthesis kit (Roche, Cat. No. 11636090910). Amplified sDNA was run on a denaturing agarose gel to confirm the size and presence of a single band. sDNA 164 was then precipitated with 0.2 M NaCl and ethanol and resuspended in 250 ?l hybridization buffer. The probe was boiled for 1hr and stored at ?20 0 C until use. The probe concentration was determined by dot-blotting on to a nitrocelulose membrane along with a marker of known concentration. The probe was detected using colorimetric asay with alkaline phosphatase-conjugated anti-DIG antibody and NBT/BCIP substrate. 1:5000 dilution of both the sense and anti-sense probes showed equivalent staining as 10 pg of a control DIG labeled fragment. Prior to use, each probe was diluted 1:2 with the hybridization buffer and boiled for 5 minutes. 5.2.4.1.3 Gonad fixation and hybridization: In situ hybridization was caried out acording to the protocol by Min-Ho Le and Tim Schedl (http:/ww.ormbook.org/toc_wormethods.html) with minor modifications. After fixation, the gonads were treated with Proteinase K for 1hr at the concentration of 100 ?g/ml of PBT. Hybridization with the DIG labeled sDNA probe was caried out at 48 0 C for 36 hrs. Subsequent washes were also caried out at 48 0 C. After blocking with BSA, the DIG probe was detected by colorimetric asay; Alkaline Phosphatase conjugated anti-DIG antibody and NBT/BCIP substrate. The gonads were incubated overnight at 4 0 C with 1:2500 diluted Alkaline Phosphatase conjugated to anti- DIG antibody and the staining was done at room temperature for 2 hrs with NBT/BCIP and 100 ng DAPI in staining solution. Finaly, gonads were mounted on an agar pad in PBS with 100 ng DAPI and the images were taken with Olympus BX51 microscope at 40X under DIC and DAPI filter. 165 5.2.4.2 Results About 5% of the elt-1 RNAi treated L2/L3 hermaphrodites lay unfertilized oocytes after reaching adulthood. Unfertilized oocytes is a characteristic of the Spe (Sperm-specific sterility) phenotype in C. elegans. Also, elt-1 is reported to be overexpresed in the sperm-producing germline (Reinke et al., 2000). This information points towards a sperm-specific role of elt-1. If it does play functional role in spermatogenesis, it should be expresed in the germline during sperm development. Based on the signal patern of the DIG-labeled anti-sense probe, elt-1 is indeed expresed in the meiotic region of spermatogenic germline (Figure 5-8). fem-3(q20) hermaphrodites produce sperm throughout their life and expres elt-1 in meiotic germline at both L4 and adult stages. fem-1(hc17) adult hermaphrodites, which produce only oocytes, did not show any expresion in the meiotic germline. N2 hermaphrodites during L4 larval stage and N2 males also show elt-1 expresion in the meiotic region of the gonad (data not shown). The oocytes and sperm show non-specific staining with both sense and anti-sense probes. 166 Figure 5-8: In situ hybridization to detect elt-1 expresion in the germlines DIC images of the gonads showing anti-sense and sense elt-1 probe hybridization at 40X magnification in fem-1 and fem-3 disected gonads. Anti-sense probe showed signal only in the fem-3 gonads (top left) during sperm diferentiation phase. 167 5.2.5 Complementation test between elt-1(zu180) and it129 ts . 5.2.5.1 Methods The complementation analysis was done betwen two aleles, elt-1(zu180) and it129 ts , as described in the following figure (Figure 5-9). Figure 5-9: Strategy used to generate heterozygous elt-1(zu180) over (it129 ts ) worms. it129 ts dpy-20 elt-1 unc-43 P0s crossed at L3 at 15 0 C it129 ts dpy-20 dpy-20 F1: it129 ts dpy-20 it129 ts dpy-20 F1s shifted to 25 0 C as L3s elt-1 unc-43 dpy-20 WT DPY 168 5.2.5.2 Results F1 progeny from the cross as described in Figure 5-9 is expected to give 50% wild type and 50% dumpy worms. Both wild type and dumpy worms were observed in the progeny with 1:1 ratio of hermaphrodites to males. Al of the heterozygous it129 ts /elt- 1(zu180) tested (20 hermaphrodites from each cros) laid viable progeny. Both the aleles complement each other for embryonic lethality and sperm-specific sterility. Thus, it129 ts is not an alele of elt-1(zu180). As described in chapter I, it129 ts alele encodes a point mutation in uba-1 (ubiquitin-activating enzyme) gene. 25% of the total progeny from the cross (Figure 5-9) was recorded as dead embryos. A few of the non-dumpy F1 hermaphrodites which are heterozygous for both, it129 ts and elt-1(zu180) aleles, showed partial male like tail development (?intersex? phenotype as it wil be refered to hereafter) as shown in Figure 5-10. The ?intersex? phenotype is observed at low penetrance but persists in the hermaphrodites throughout their life. Thus, elt-1 and it129 ts show genetic interaction in the double heterozygous condition. Additional experiments were undertaken to further characterize the interaction. 5.2.6 elt-1 RNAi or wild type elt-1 genomic region microinjection in uba-1(it129 ts ) hermaphrodites leads to ?intersex? phenotype. 5.2.6.1 Methods elt-1 RNAi was performed on it129 ts L2 hermaphrodites grown at 15 0 C as described in 5.2.2.1 and the adult hermaphrodites were scored for ?intersex? phenotype. The elt-1 gene was also introduced into uba-1(it129 ts ) strain by micro-injection. 169 Genomic region of 10Kb ecoding elt-1 gene was PCR amplified from wild type genomic DNA and was purified by precipitating with potasium acetate and ethanol. Injection mix was prepared with 100 ng elt-1 PCR DNA, 450 ng of EcoRI digested pRF4 (rol-6 transgenic marker) and 200 ng of PuvII digested wild type genomic DNA to the final concentration of 156 ng/?l. The mix was injected in homozygous it129 ts hermaphrodites. The hermaphrodites and the progeny were maintained at 15 0 C. Transgenic lines were isolated based on the roller phenotype confered by rol-6 marker. 5.2.6.2 Results By performing elt-1 RNAi on it129 ts worms at 25 0 C, elt-1 activity was reduced in the uba-1 mutant background where both (uba-1 and elt-1) the gene functions are reduced. The intersex phenotype was reproduced in the F1 hermaphrodites at low frequency. About 2 in 30 hermaphrodites had intersex tail. The tail retraction persisted throughout the life as sen in the double heterozygous it129 ts /elt-1(zu180) hermaphrodites. More frequent (10 in 30 transgenic worms) but transient intersex phenotype was observed in it129 ts transgenic hermaphrodites expresing elt-1 transgene. It is possible that overexpresion of elt-1 mRNA in the transgenic lines is leading to co-suppresion of elt-1 (Keting and Plasterk, 2000). During co-suppresion, overexpresed transcript of the gene from the transgene leads to (as speculated, smal RNAs mediated) degradation of itself along with the native transcript from the homologous gene. As a result, the 170 Figure 5-10: Intersex phenotype in transgenic uba-1(it129 ts ) hermaphrodites carrying elt-1 transgene. Arow indicates the male like developing tale in the hermaprhodite soma of L3 stage. The intersex tale is transient as normal hermaphrodite tale appears by adulthood (top left of the image). 171 stransgenic lines have reduced or no expresion of elt-1 in it129 ts mutant background, creating similar phenotype as observed in it129 ts / elt-1 double heterozygotes. It is also likely that ELT-1 itself is targeted for ubiquitin-mediated degradation, which is inhibited in the uba-1 mutant background leading to observed abnormalities. Male tail is specialized organ for copulatory functions performed by the male. The development of the tail is acheived by the retraction of the cels in the tail region of the male during L4 larval stage. This retraction does not occur in hermaphrodites as a result the hermaphrodite tail develops with a tapering end. In ?intersex? hermaphrodites where expresion of both the genes; elt-1 and uba-1 is reduced, the tail retraction is possibly activated in hermaphrodites during larval stages giving rise to L4 male tail structure. Where as, in the transgenic worms, co-suppresion could occur transiently leading to transient ?intersex? phenotype. 172 BIBLIOGRAPHY Al-Hakim, A. K., Zagorska, A., Chapman, L., Deak, M., Peggie, M., and Alesi, D. R. (2008). Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)- linked polyubiquitin chains. Biochem J 411, 249-260. Ardley, H. C., and Robinson, P. A. (2004). The role of ubiquitin-protein ligases in neurodegenerative disease. Neurodegener Dis 1, 71-87. Argon, Y., and Ward, S. (1980). Caenorhabditis elegans fertilization-defective mutants with abnormal sperm. Genetics 96, 413-433. Ayusawa, D., Kaneda, S., Itoh, Y., Yasuda, H., Murakami, Y., Sugasawa, K., Hanaoka, F., and Seno, T. (1992). Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arested mouse FM3A cel mutant with thermolabile E1. Cel Struct Funct 17, 113-122. Barends, W. M., Hoogerbrugge, J. W., Roest, H. P., Ooms, M., Vreburg, J., Hoeijmakers, J. H., and Grootegoed, J. A. (1999a). Histone ubiquitination and chromatin remodeling in mouse spermatogenesis. Dev Biol 207, 322-333. Barends, W. M., Roest, H. P., and Grootegoed, J. A. (1999b). The ubiquitin system in gametogenesis. Mol Cel Endocrinol 151, 5-16. Barends, W. M., van der Lan, R., and Grootegoed, J. A. (2000). Specific aspects of the ubiquitin system in spermatogenesis. J Endocrinol Invest 23, 597-604. Baird, S. E., and Elazar, S. A. (1999). TGFbeta-like signaling and spicule development in Caenorhabditis elegans. Dev Biol 212, 93-100. Barton, M. K., and Kimble, J. (1990). fog-1, a regulatory gene required for specification of spermatogenesis in the germ line of Caenorhabditis elegans. Genetics 125, 29-39. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Gain-of-function mutations of fem- 3, a sex-determination gene in Caenorhabditis elegans. Genetics 115, 107-119. 173 Batchelder, C., Dunn, M. A., Choy, B., Suh, Y., Casie, C., Shim, E. Y., Shin, T. H., Melo, C., Seydoux, G., and Blackwel, T. K. (1999). Transcriptional represion by the Caenorhabditis elegans germ-line protein PIE-1. Genes Dev 13, 202-212. Baugh, L. R., Hil, A. A., Clagget, J. M., Hil-Harfe, K., Wen, J. C., Slonim, D. K., Brown, E. L., and Hunter, C. P. (2005). The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo. Development 132, 1843-1854. Baugh, L. R., Hil, A. A., Slonim, D. K., Brown, E. L., and Hunter, C. P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900. Beanan, M. J., and Strome, S. (1992). Characterization of a germ-line proliferation mutation in C. elegans. Development 116, 755-766. Ben-Sadon, R., Zaroor, D., Ziv, T., and Ciechanover, A. (2006). The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity. Mol Cel 24, 701-711. Bery, L. W., Westlund, B., and Schedl, T. (1997). Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors. Development 124, 925-936. Bloch, D. B., de la Monte, S. M., Guigaouri, P., Filippov, A., and Bloch, K. D. (1996). Identification and characterization of a leukocyte-specific component of the nuclear body. J Biol Chem 271, 29198-29204. Bloch, D. B., Nakajima, A., Gulick, T., Chiche, J. D., Orth, D., de La Monte, S. M., and Bloch, K. D. (2000). Sp110 localizes to the PML-Sp100 nuclear body and may function as a nuclear hormone receptor transcriptional coactivator. Mol Cel Biol 20, 6138-6146. Bosu, D. R., and Kipreos, E. T. (2008). Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cel Div 3, 7. 174 Bottomley, M. J., Collard, M. W., Huggenvik, J. I., Liu, Z., Gibson, T. J., and Satler, M. (2001). The SAND domain structure defines a novel DNA-binding fold in transcriptional regulation. Nat Struct Biol 8, 626-633. Bourbon, H. M., Aguilera, A., Ansari, A. Z., Asturias, F. J., Berk, A. J., Bjorklund, S., Blackwel, T. K., Borggrefe, T., Carey, M., Carlson, M., et al. (2004). A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase I. Mol Cel 14, 553-557. Bowerman, B., Draper, B. W., Melo, C. C., and Pries, J. R. (1993). The maternal gene skn-1 encodes a protein that is distributed unequaly in early C. elegans embryos. Cel 74, 443-452. Bowerman, B., Eaton, B. A., and Pries, J. R. (1992). skn-1, a maternaly expresed gene required to specify the fate of ventral blastomeres in the early C. elegans embryo. Cel 68, 1061-1075. Bowerman, B., and Kurz, T. (2006). Degrade to create: developmental requirements for ubiquitin-mediated proteolysis during early C. elegans embryogenesis. Development 133, 773-784. Boyd, S. D. (2008). Everything you wanted to know about smal RNA but were afraid to ask. Lab Invest 88, 569-578. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Buonomo, S. B., Clyne, R. K., Fuchs, J., Loidl, J., Uhlmann, F., and Nasmyth, K. (2000). Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cel 103, 387-398. Campbel, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J. (1998). Increasing complexity of Ras signaling. Oncogene 17, 1395-1413. Catic, A., and Ploegh, H. L. (2005). Ubiquitin--conserved protein or selfish gene? Trends Biochem Sci 30, 600-604. Chan, N. L., and Hil, C. P. (2001). Defining polyubiquitin chain topology. Nat Struct Biol 8, 650-652. 175 Chasnov, J. R., So, W. K., Chan, C. M., and Chow, K. L. (2007). The species, sex, and stage specificity of a Caenorhabditis sex pheromone. Proc Natl Acad Sci U S A 104, 6730-6735. Chastagner, P., Israel, A., and Brou, C. (2006). Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep 7, 1147- 1153. Chaterje, I., Richmond, A., Putiri, E., Shakes, D. C., and Singson, A. (2005). The Caenorhabditis elegans spe-38 gene encodes a novel four-pas integral membrane protein required for sperm function at fertilization. Development 132, 2795-2808. Chechi, P. M., and Kely, W. G. (2006). emb-4 is a conserved gene required for eficient germline-specific chromatin remodeling during Caenorhabditis elegans embryogenesis. Genetics 174, 1895-1906. Chen, P., and Elis, R. E. (2000). TRA-1A regulates transcription of fog-3, which controls germ cel fate in C. elegans. Development 127, 3119-3129. Chernova, T. A., Alen, K. D., Wesoloski, L. M., Shanks, J. R., Chernof, Y. O., and Wilkinson, K. D. (2003). Pleiotropic efects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the fre ubiquitin pool. J Biol Chem 278, 52102- 52115. Chiu, Y. H., Sun, Q., and Chen, Z. J. (2007). E1-L2 activates both ubiquitin and FAT10. Mol Cel 27, 1014-1023. Christensen, J., Cotmore, S. F., and Tatersal, P. (1999). Two new members of the emerging KDWK family of combinatorial transcription modulators bind as a heterodimer to flexibly spaced PuCGPy half-sites. Mol Cel Biol, 7741-7750. Ciechanover, A., Elias, S., Heler, H., and Hershko, A. (1982). "Covalent afinity" purification of ubiquitin-activating enzyme. J Biol Chem 257, 2537-2542. Ciechanover, A., Finley, D., and Varshavsky, A. (1984). Ubiquitin dependence of selective protein degradation demonstrated in the mamalian cel cycle mutant ts85. Cel 37, 57-66. 176 Ciechanover, A., Finley, D., and Varshavsky, A. (1985). Mamalian cel cycle mutant defective in intracelular protein degradation and ubiquitin-protein conjugation. Prog Clin Biol Res 180, 17-31. Ciechanover, A., and Schwartz, A. L. (1989). The ubiquitin-dependent proteolytic pathway: specificity of recognition of the proteolytic substrates. Revis Biol Celular 20, 217-234. Cohen-Fix, O., Peters, J. M., Kirschner, M. W., and Koshland, D. (1996). Anaphase initiation in Sacharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10, 3081-3093. Conradt, B., and Horvitz, H. R. (1998). The C. elegans protein EGL-1 is required for programed cel death and interacts with the Bcl-2-like protein CED-9. Cel 93, 519-529. Conradt, B., and Horvitz, H. R. (1999). The TRA-1A sex determination protein of C. elegans regulates sexualy dimorphic cel deaths by represing the egl-1 cel death activator gene. Cel 98, 317-327. Cook, J. C., and Chock, P. B. (1992). Isoforms of mamalian ubiquitin-activating enzyme. J Biol Chem 267, 24315-24321. Cook, J. C., and Chock, P. B. (1995). Phosphorylation of ubiquitin-activating enzyme in cultured cels. Proc Natl Acad Sci U S A 92, 3454-3457. Davis, E. S., Wile, L., Chestnut, B. A., Sadler, P. L., Shakes, D. C., and Golden, A. (2002). Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I. Genetics 160, 805-813. Del Rio-Albrechtsen, T., Kiontke, K., Chiou, S. Y., and Fitch, D. H. (2006). Novel gain- of-function aleles demonstrate a role for the heterochronic gene lin-41 in C. elegans male tail tip morphogenesis. Dev Biol 297, 74-86. DeRenzo, C., Rese, K. J., and Seydoux, G. (2003). Exclusion of germ plasm proteins from somatic lineages by cullin-dependent degradation. Nature 424, 685-689. 177 Ding, M., Chao, D., Wang, G., and Shen, K. (2007). Spatial regulation of an E3 ubiquitin ligase directs selective synapse elimination. Science 317, 947-951. Draper, B. W., Melo, C. C., Bowerman, B., Hardin, J., and Pries, J. R. (1996). MEX-3 is a KH domain protein that regulates blastomere identity in early C. elegans embryos. Cel 87, 205-216. Dupuy, D., Bertin, N., Hidalgo, C. A., Venkatesan, K., Tu, D., Le, D., Rosenberg, J., Svrzikapa, N., Blanc, A., Carnec, A., et al. (2007). Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans. Nat Biotechnol 25, 663-668. Eletr, Z. M., Huang, D. T., Duda, D. M., Schulman, B. A., and Kuhlman, B. (2005). E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat Struct Mol Biol 12, 933-934. Elis, R. E., and Kimble, J. (1995). The fog-3 gene and regulation of cel fate in the germ line of Caenorhabditis elegans. Genetics 139, 561-577. Finley, D., Ciechanover, A., and Varshavsky, A. (1984). Thermolability of ubiquitin- activating enzyme from the mamalian cel cycle mutant ts85. Cel 37, 43-55. Ford, L. P., Bagga, P. S., and Wilusz, J. (1997). The poly(A) tail inhibits the asembly of a 3'-to-5' exonuclease in an in vitro RNA stability system. Mol Cel Biol, 398-406. Francis, R., Barton, M. K., Kimble, J., and Schedl, T. (1995). gld-1, a tumor suppresor gene required for oocyte development in Caenorhabditis elegans. Genetics 139, 579-606. Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325-330. Frazier, T., Shakes, D., Hota, U., and Boyd, L. (2004). Caenorhabditis elegans UBC-2 functions with the anaphase-promoting complex but also has other activities. J Cel Sci 117, 5427-5435. 178 Fukushige, T., Brodigan, T. M., Schriefer, L. A., Waterston, R. H., and Krause, M. (2006). Defining the transcriptional redundancy of early bodywal muscle development in C. elegans: evidence for a unified theory of animal muscle development. Genes Dev 20, 3395-3406. Funabiki, H., Yamano, H., Kumada, K., Nagao, K., Hunt, T., and Yanagida, M. (1996). Cut2 proteolysis required for sister-chromatid seperation in fision yeast. Nature 381, 438-441. Furuta, T., Tuck, S., Kirchner, J., Koch, B., Auty, R., Kitagawa, R., Rose, A. M., and Grenstein, D. (2000). EMB-30: an APC4 homologue required for metaphase-to- anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Mol Biol Cel 11, 1401-1419. Getha, T., Kenchappa, R. S., Wooten, M. W., and Carter, B. D. (2005). TRAF6- mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor interactor. Embo J 24, 3859-3868. Ghaboosi, N., and Deshaies, R. J. (2007). A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome. Mol Biol Cel 18, 1953-1963. Ghosh, D., and Seydoux, G. (2008). Inhibition of transcription by the Caenorhabditis elegans germline protein PIE-1: genetic evidence for distinct mechanisms targeting initiation and elongation. Genetics 178, 235-243. Gibson, T. J., Ramu, C., Gemund, C., and Aasland, R. (1998). The APECED polyglandular autoimune syndrome protein, AIRE-1, contains the SAND domain and is probably a transcription factor. Trends Biochem Sci 23, 242-244. Gileard, J. S., and McGhee, J. D. (2001). Activation of hypodermal diferentiation in the Caenorhabditis elegans embryo by GATA transcription factors ELT-1 and ELT-3. Mol Cel Biol 21, 2533-2544. Gileard, J. S., Shafi, Y., Bary, J. D., and McGhee, J. D. (1999). ELT-3: A Caenorhabditis elegans GATA factor expresed in the embryonic epidermis during morphogenesis. Dev Biol 208, 265-280. 179 Gladden, J. M., and Meyer, B. J. (2007). A ONECUT Homeodomain Protein Communicates X Chromosome Dose to Specify Caenorhabditis elegans Sexual Fate by Represing a Sex Switch Gene. Genetics 177, 1621-1637. Golden, A., Sadler, P. L., Walenfang, M. R., Schumacher, J. M., Hamil, D. R., Bates, G., Bowerman, B., Seydoux, G., and Shakes, D. C. (2000). etaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans. J Cel Biol 151, 1469- 1482. Gonczy, P., and Rose, L. S. Asymmetric cel division and axis formation in the embryo, In WormBook, T. C. e. R. Community, ed. (WormBook). Graham, R. W., Jones, D., and Candido, E. P. (1989). UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expresed. Mol Cel Biol 9, 268-277. Gross, C. T., and McGinnis, W. (1996). DEAF-1, a novel protein that binds an esential region in a Deformed response element. EMBO J 15, 1961-1970. Guardavacaro, D., Kudo, Y., Boulaire, J., Barchi, M., Busino, L., Donzeli, M., Margottin-Goguet, F., Jackson, P. K., Yamasaki, L., and Pagano, M. (2003). Control of meiotic and mitotic progresion by the F box protein beta-Trcp1 in vivo. Dev Cel 4, 799-812. Gudgen, M., Chandrasekaran, A., Frazier, T., and Boyd, L. (2004). Interactions within the ubiquitin pathway of Caenorhabditis elegans. Biochem Biophys Res Commun 325, 479-486. Guedes, S., and Pries, J. R. (1997). The C. elegans MEX-1 protein is present in germline blastomeres and is a P granule component. Development 124, 731-739. Has, A. L., and Rose, I. A. (1982). The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 257, 10329-10337. Haglund, K., and Dikic, I. (2005). Ubiquitylation and cel signaling. Embo J 24, 3353- 3359. 180 Haglund, K., Sigismund, S., Polo, S., Szymkiewicz, I., Di Fiore, P. P., and Dikic, I. (2003). Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cel Biol 5, 461-466. Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994). Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs. J Biol Chem 269, 33171-33178. Hanna, J., and Finley, D. (2007). A proteasome for al occasions. FEBS Let 581, 2854- 2861. Hanna, J., Legget, D. S., and Finley, D. (2003). Ubiquitin depletion as a key mediator of toxicity by translational inhibitors. Mol Cel Biol 23, 9251-9261. Hanna, J., Meides, A., Zhang, D. P., and Finley, D. (2007). A ubiquitin stres response induces altered proteasome composition. Cel 129, 747-759. Hansen, D., and Pilgrim, D. (1999). Sex and the single worm: sex determination in the nematode C. elegans. Mech Dev 83, 3-15. Hatfield, P. M., Calis, J., and Vierstra, R. D. (1990). Cloning of ubiquitin activating enzyme from wheat and expresion of a functional protein in Escherichia coli. J Biol Chem 265, 15813-15817. Hatfield, P. M., Gosink, M. M., Carpenter, T. B., and Vierstra, R. D. (1997). The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana. Plant J 11, 213-226. Hatfield, P. M., and Vierstra, R. D. (1992). Multiple forms of ubiquitin-activating enzyme E1 from wheat. Identification of an esential cysteine by in vitro mutagenesis. J Biol Chem 267, 14799-14803. Hershko, A. (1991). The ubiquitin pathway for protein degradation. Trends Biochem Sci 16, 265-268. 181 Hershko, A., Heler, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin- protein ligase system. Resolution, afinity purification, and role in protein breakdown. J Biol Chem 258, 8206-8214. Hicke, L. (2001). Protein regulation by monoubiquitin. Nat Rev Mol Cel Biol 2, 195- 201. Hicke, L., Schubert, H. L., and Hil, C. P. (2005). Ubiquitin-binding domains. Nat Rev Mol Cel Biol 6, 610-621. Hil, K. L., and L'Hernault, S. W. (2001). Analyses of reproductive interactions that occur after heterospecific matings within the genus Caenorhabditis. Dev Biol 232, 105- 114. Hirsh, D., Oppenheim, D., and Klas, M. (1976). Development of the reproductive system of Caenorhabditis elegans. Dev Biol 49, 200-219. Hirsh, D., and Vanderslice, R. (1976). Temperature-sensitive developmental mutants of Caenorhabditis elegans. Dev Biol 49, 220-235. Hochstraser, M. (2000). Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cel Biol 2, E153-157. Hodges, M., Tisot, C., Howe, K., Grimwade, D., and Fremont, P. S. (1998). Structure, organization, and dynamics of promyelocytic leukemia protein nuclear bodies. Am J Hum Genet 63, 297-304. Hodgkin, J. (1986). Sex determination in the nematode C. elegans: analysis of tra-3 suppresors and characterization of fem genes. Genetics 114, 15-52. Hodgkin, J. (1987). A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, 731-745. Hodgkin, J., Horvitz, H. R., and Brenner, S. (1979). Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS. Genetics 91, 67-94. 182 Hodgkin, J. A., and Brenner, S. (1977). Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans. Genetics 86, 275-287. Hoppe, T., Casata, G., Baral, J. M., Springer, W., Hutagalung, A. H., Epstein, H. F., and Baumeister, R. (2004). Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans. Cel 118, 337-349. Howard, R. A., Sharma, P., Hajar, C., Caldwel, K. A., Caldwel, G. A., du Breuil, R., Moore, R., and Boyd, L. (2007). Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation. BMC el Biol 8, 32. Huang, D. T., Paydar, A., Zhuang, M., Waddel, M. B., Holton, J. M., and Schulman, B. A. (2005). Structural basis for recruitment of Ubc12 by an E2 binding domain in NED8's E1. Mol Cel 17, 341-350. Huggenvik, J. I., Michelson, R. J., Collard, M. W., Ziemba, A. J., Gurley, P., and Mowen, K. A. (1998). Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein. Mol Endocrinol, 1619-1639. Hunter, C. P., and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cel 87, 217-226. Hunter, C. P., and Wood, W. B. (1992). Evidence from mosaic analysis of the masculinizing gene her-1 for cel interactions in C. elegans sex determination. Nature 355, 551-555. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., et al. (2000). A ubiquitin- like system ediates protein lipidation. Nature 408, 488-492. Itoh, S., and ten Dijke, P. (2007). Negative regulation of TGF-beta receptor/Smad signal transduction. Curr Opin Cel Biol 19, 176-184. Jackson, P. K., Eldridge, A. G., Fred, E., Furstenthal, L., Hsu, J. Y., Kaiser, B. K., and Reimann, J. D. (2000). The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cel Biol 10, 429-439. 183 Jager, S., Schwartz, H. T., Horvitz, H. R., and Conradt, B. (2004). The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM- 1 and FEM-3 for degradation by the proteasome. Proc Natl Acad Sci U S A 101, 12549-12554. Jentsch, S. (1992). The ubiquitin-conjugation system. Annu Rev Genet 26, 179-207. Jentsch, S., and Pyrowolakis, G. (2000). Ubiquitin and its kin: how close are the family ties? Trends Cel Biol 10, 335-342. Jentsch, S., Seufert, W., and Hauser, H. P. (1991). Genetic analysis of the ubiquitin system. Biochim Biophys Acta 1089, 127-139. Jiang, M., Ryu, J., Kiraly, M., Duke, K., Reinke, V., and Kim, S. K. (2001). Genome- wide analysis of developmental and sex-regulated gene expresion profiles in Caenorhabditis elegans. Proc Natl Acad Sci U S A 98, 218-223. Jin, S. W., Arno, N., Cohen, A., Shah, A., Xu, Q., Chen, N., and Elis, R. E. (2001). In Caenorhabditis elegans, the RNA-binding domains of the cytoplasmic polyadenylation element binding protein FOG-1 are needed to regulate germ cel fates. Genetics 159, 1617-1630. Johnson, E. S. (2004). Protein modification by SUMO. Annu Rev Biochem 73, 355-382. Johnston, S. C., Riddle, S. M., Cohen, R. E., and Hil, C. P. (1999). Structural basis for the specificity of ubiquitin C-terminal hydrolases. Embo J 18, 3877-3887. Jones, D., and Candido, E. P. (1993). Novel ubiquitin-like ribosomal protein fusion genes from the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. J Biol Chem 268, 19545-19551. Jones, D., Crowe, E., Stevens, T. A., and Candido, E. P. (2002). Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins. Genome Biol 3, RESEARCH0002. Jones, S. J., Riddle, D. L., Pouzyrev, A. T., Velculescu, V. E., Hilier, L., Eddy, S. R., Stricklin, S. L., Bailie, D. L., Waterston, R., and Mara, M. A. (2001). Changes in 184 gene expresion asociated with developmental arest and longevity in Caenorhabditis elegans. Genome Res 11, 1346-1352. Juo, P., and Kaplan, J. M. (2004). The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans. Curr Biol 14, 2057-2062. Kaiser, P., Flick, K., Witenberg, C., and Red, S. I. (2000). Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4. Cel 102, 303-314. Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231-237. Kawakami, T., Chiba, T., Suzuki, T., Iwai, K., Yamanaka, K., Minato, N., Suzuki, H., Shimbara, N., Hidaka, Y., Osaka, F., et al. (2001). NED8 recruits E2-ubiquitin to SCF E3 ligase. Embo J 20, 4003-4012. Kay, G. F., Ashworth, A., Penny, G. D., Dunlop, M., Swift, S., Brockdorf, N., and Rastan, S. (1991). A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1. Nature 354, 486-489. Ke, Y., and Huibregtse, J. M. (2007). Regulation of catalytic activities of HECT ubiquitin ligases. Biochem Biophys Res Commun 354, 329-333. Keleher, J. F., Mandel, M. A., Moulder, G., Hil, K. L., L'Hernault, S. W., Barstead, R., and Titus, . A. (2000). yosin VI is required for asymmetric segregation of celular components during C. elegans spermatogenesis. Curr Biol 10, 1489-1496. Kemphues, K. J., Wolf, N., Wood, W. B., and Hirsh, D. (1986). Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans. Dev Biol 113, 449-460. Kerscher, O., Felberbaum, R., and Hochstraser, M. (2006). Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cel Dev Biol 22, 159-180. 185 Keting, R. F., and Plasterk, R. H. (2000). A genetic link betwen co-suppresion and RNA interference in C. elegans. Nature 404, 296-298. Kim, E. Y., Ridgway, L. D., Zou, S., Chiu, Y. H., and Dryer, S. E. (2007a). Alternatively spliced C-terminal domains regulate the surface expresion of large conductance calcium-activated potasium channels. Neuroscience 146, 1652-1661. Kim, H. T., Kim, K. P., Lledias, F., Kiselev, A. F., Scaglione, K. M., Skowyra, D., Gygi, S. P., and Goldberg, A. L. (2007b). Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing al possible isopeptide linkages. J Biol Chem 282, 17375-17386. Kim, M. O., Chawla, P., Overland, R. P., Xia, E., Sadri-Vakili, G., and Cha, J. H. (2008). Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation. J Neurosci 28, 3947-3957. Kim, S. K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J. M., Eizinger, A., Wylie, B. N., and Davidson, G. S. (2001). A gene expresion map for Caenorhabditis elegans. Science 293, 2087-2092. Kim, V. N. (2005). Smal RNAs: clasification, biogenesis, and function. Mol Cels 19, 1-15. Kimble, J. (1988). Genetic control of sex determination in the germ line of Caenorhabditis elegans. Philos Trans R Soc Lond B iol Sci 322, 11-18. Kipreos, E. T. Ubiquitin-mediated pathways in C. elegans, In WormBook, T. C. e. R. Community, ed. (WormBook). Kitagawa, R., Law, E., Tang, L., and Rose, A. M. (2002). The Cdc20 homolog, FZY-1, and its interacting protein, IFY-1, are required for proper chromosome segregation in Caenorhabditis elegans. Curr Biol 12, 2118-2123. Kosinski, M., McDonald, K., Schwartz, J., Yamamoto, I., and Grenstein, D. (2005). C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes. Development 132, 3357-3369. 186 Krause, M., Harison, S. W., Xu, S. Q., Chen, L., and Fire, A. (1994). Elements regulating cel- and stage-specific expresion of the C. elegans MyoD family homolog hlh-1. Dev Biol 166, 133-148. Kroft, T. L., Gleason, E. J., and L'Hernault, S. W. (2005). The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein. Dev Biol 286, 169-181. Kulka, R. G., Raboy, B., Schuster, R., Parag, H. A., Diamond, G., Ciechanover, A., and Marcus, M. (1988). A Chinese hamster cel cycle mutant arested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1. J Biol Chem 263, 15726- 15731. Kumar, P. G., Laloraya, M., Wang, C. Y., Ruan, Q. G., Davoodi-Semiromi, A., Kao, K. J., and She, J. X. (2001). The autoimune regulator (AIRE) is a DNA-binding protein. J Biol Chem 276, 41357-41364. L'Hernault, S. W. Spermatogenesis, In WormBook, T. C. e. R. Community, ed. (WormBook). L'Hernault, S. W., and Arduengo, P. M. (1992). Mutation of a putative sperm embrane protein in Caenorhabditis elegans prevents sperm diferentiation but not its asociated meiotic divisions. J Cel Biol 119, 55-68. L'Hernault, S. W., Shakes, D. C., and Ward, S. (1988). Developmental genetics of chromosome I spermatogenesis-defective mutants in the nematode Caenorhabditis elegans. Genetics 120, 435-452. Labouese, M., Hartwieg, E., and Horvitz, H. R. (1996). The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain al non-neuronal ectodermal cel fates. Development 122, 2579-2588. Lamitina, S. T., and L'Hernault, S. W. (2002). Dominant mutations in the Caenorhabditis elegans Myt1 ortholog we-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis. Development 129, 5009-5018. 187 Lamont, L. B., and Kimble, J. (2007). Developmental expresion of FOG-1/CPEB protein and its control in the Caenorhabditis elegans hermaphrodite germ line. Dev Dyn 236, 871-879. LaMunyon, C. W., and Ward, S. (1998). Larger sperm outcompete smaler sperm in the nematode Caenorhabditis elegans. Proc Biol Sci 265, 1997-2002. Le, M.-H., and Schedl, T. RNA in situ hybridization of disected gonads, In WormBook, T. C. e. R. Community, ed. (WormBook). Le, T. I., and Young, R. A. (1998). Regulation of gene expresion by TBP-asociated proteins. Genes Dev 12, 1398-1408. Le, T. V., Ding, T., Chen, Z., Rajendran, V., Scher, H., Lackey, M., Bolduc, C., and Bergmann, A. (2008). The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tisue growth non-autonomously. Development 135, 43-52. Legget, D. S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M., Baker, R. T., Walz, T., Ploegh, H., and Finley, D. (2002). Multiple asociated proteins regulate proteasome structure and function. Mol Cel 10, 495-507. Li, W., and Ye, Y. (2008). Polyubiquitin chains: functions, structures, and mechanisms. Cel Mol Life Sci. Liao, E. H., Hung, W., Abrams, B., and Zhen, M. (2004). An SCF-like ubiquitin ligase complex that controls presynaptic diferentiation. Nature 430, 345-350. Lin, R. (2003). A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation, results in delayed degradation of maternal proteins and embryonic lethality. Dev Biol 258, 226-239. Liu, J., Vasudevan, S., and Kipreos, E. T. (2004). CUL-2 and ZYG-11 promote meiotic anaphase I and the proper placement of the anterior-posterior axis in C. elegans. Development 131, 3513-3525. Lois, L. M., and Lima, C. D. (2005). Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1. Embo J 24, 439-451. 188 Luitjens, C., Galegos, M., Kraemer, B., Kimble, J., and Wickens, M. (2000). CPEB proteins control two key steps in spermatogenesis in C. elegans. Genes Dev 14, 2596-2609. Luz, J. G., Hasig, C. A., Pickle, C., Godzik, A., Meyer, B. J., and Wilson, I. A. (2003). XOL-1, primary determinant of sexual fate in C. elegans, is a GHMP kinase family member and a structural prototype for a clas of developmental regulators. Genes Dev 17, 977-990. Machaca, K., DeFelice, L. J., and L'Hernault, S. W. (1996). A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid diferentiation. Dev Biol 176, 1-16. Madl, J. E., and Herman, R. K. (1979). Polyploids and sex determination in Caenorhabditis elegans. Genetics 93, 393-402. Maduro, M. F., Meneghini, M. D., Bowerman, B., Broitman-Maduro, G., and Rothman, J. H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol Cel 7, 475-485. Martinho, R. G., Kunwar, P. S., Casanova, J., and Lehmann, R. (2004). A noncoding RNA is required for the represion of RNApolI-dependent transcription in primordial germ cels. Curr Biol 14, 159-165. McGrath, J. P., Jentsch, S., and Varshavsky, A. (1991). UBA 1: an esential yeast gene encoding ubiquitin-activating enzyme. Embo J 10, 227-236. McKay, J. P., Raizen, D. M., Gottschalk, A., Schafer, W. R., and Avery, L. (2004). eat-2 and eat-18 are required for nicotinic neurotransmision in the Caenorhabditis elegans pharynx. Genetics 166, 161-169. Mehta, N., Loria, P. M., and Hobert, O. (2004). A genetic scren for neurite outgrowth mutants in Caenorhabditis elegans reveals a new function for the F-box ubiquitin ligase component LIN-23. Genetics 166, 1253-1267. 189 Melo, C. C., Kramer, J. M., Stinchcomb, D., and Ambros, V. (1991). Eficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J 10, 3959-3970. Melo, C. C., Schubert, C., Draper, B., Zhang, W., Lobel, R., and Pries, J. R. (1996). The PIE-1 protein and germline specification in C. elegans embryos. Nature 382, 710- 712. Miranda, T. B., and Jones, P. A. (2007). DNA methylation: the nuts and bolts of represion. J Cel Physiol 213, 384-390. Mitchel, M. J., Woods, D. R., Wilcox, S. A., Graves, J. A., and Bishop, C. E. (1992). Marsupial Y chromosome encodes a homologue of the mouse Y-linked candidate spermatogenesis gene Ube1y. Nature 359, 528-531. Morokuma, Y., Nakamura, N., Kato, A., Notoya, M., Yamamoto, Y., Sakai, Y., Fukuda, H., Yamashina, S., Hirata, Y., and Hirose, S. (2007). MARCH-XI, a novel transmembrane ubiquitin ligase implicated in ubiquitin-dependent protein sorting in developing spermatids. J Biol Chem 282, 24806-24815. Nair, R., Carter, P., and Rost, B. (2003). NLSdb: database of nuclear localization signals. Nucleic Acids Res 31, 397-399. Nakayama, K. I., and Nakayama, K. (2006). Ubiquitin ligases: cel-cycle control and cancer. Nat Rev Cancer 6, 369-381. Nasmyth, K., Peters, J. M., and Uhlmann, F. (2000). Spliting the chromosome: cutting the ties that bind sister chromatids. Science 288, 1379-1385. Nelson, G. A., Lew, K. K., and Ward, S. (1978). Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Dev Biol 66, 386-409. Nelson, G. A., and Ward, S. (1980). Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin. Cel 19, 457-464. Nenoi, M., Ichimura, S., and Mita, K. (2000). Interspecific comparison in the frequency of concerted evolution at the polyubiquitin gene locus. J Mol Evol 51, 161-165. 190 Odorisio, T., Mahadevaiah, S. K., McCarey, J. R., and Burgoyne, P. S. (1996). Transcriptional analysis of the candidate spermatogenesis gene Ube1y and of the closely related Ube1x shows that they are coexpresed in spermatogonia and spermatids but are represed in pachytene spermatocytes. Dev Biol 180, 336-343. Okazaki, K., Okayama, H., and Niwa, O. (2000). The polyubiquitin gene is esential for meiosis in fision yeast. Exp Cel Res 254, 143-152. Ozkaynak, E., Finley, D., and Varshavsky, A. (1984). The yeast ubiquitin gene: head-to- tail repeats encoding a polyubiquitin precursor protein. Nature 312, 663-666. Page, B. D., Diede, S. J., Tenlen, J. R., and Ferguson, E. L. (2007). EL-1, a Hect E3 ubiquitin ligase, controls asymmetry and persistence of the SKN-1 transcription factor in the early C. elegans embryo. Development 134, 2303-2314. Page, B. D., Zhang, W., Steward, K., Blumenthal, T., and Pries, J. R. (1997). ELT-1, a GATA-like transcription factor, is required for epidermal cel fates in Caenorhabditis elegans embryos. Genes Dev 11, 1651-1661. Park, Y., Moon, Y., and Chung, H. Y. (2003). AIRE-1 (autoimune regulator type 1) as a regulator of the thymic induction of negative selection. Ann N Y Acad Sci 1005, 431-435. Pery, M. D., Li, W., Trent, C., Robertson, B., Fire, A., Hageman, J. M., and Wood, W. B. (1993). Molecular characterization of the her-1 gene suggests a direct role in cel signaling during Caenorhabditis elegans sex determination. Genes Dev 7, 216-228. Petroski, M. D., and Deshaies, R. J. (2005). Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cel Biol 6, 9-20. Pickart, C. M., and Rose, I. A. (1985). Functional heterogeneity of ubiquitin carier proteins. J Biol Chem 260, 1573-1581. Pintard, L., Wilis, J. H., Wilems, A., Johnson, J. L., Srayko, M., Kurz, T., Glaser, S., Mains, P. E., Tyers, M., Bowerman, B., and Peter, M. (2003). The BTB protein EL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425, 311-316. 191 Pique, M., Lopez, J. M., Foisac, S., Guigo, R., and Mendez, R. (2008). A combinatorial code for CPE-mediated translational control. Cel 132, 434-448. Powel, J. R., Jow, M. M., and Meyer, B. J. (2005). The T-box transcription factor SEA-1 is an autosomal element of the X:A signal that determines C. elegans sex. Dev Cel 9, 339-349. Rappleye, C. A., Tagawa, A., Lyczak, R., Bowerman, B., and Aroian, R. V. (2002). The anaphase-promoting complex and separin are required for embryonic anterior- posterior axis formation. Dev Cel 2, 195-206. Rechsteiner, M. (1987). Ubiquitin-mediated pathways for intracelular proteolysis. Annu Rev Cel Biol 3, 1-30. Rece-Hoyes, J. S., Deplancke, B., Shingles, J., Grove, C. A., Hope, I. A., and Walhout, A. J. (2005). A compendium of Caenorhabditis elegans regulatory transcription factors: a resource for mapping transcription regulatory networks. Genome Biol 6, R110. Reinke, V., Gil, I. S., Ward, S., and Kazmer, K. (2004). Genome-wide germline-enriched and sex-biased expresion profiles in Caenorhabditis elegans. Development 131, 311-323. Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S., and Kim, S. K. (2000). A global profile of germline gene expresion in C. elegans. Mol Cel 6, 605-616. Reis, Y., Heler, H., and Hershko, A. (1989). Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carier protein. J Biol Chem 264, 10378-10383. Richter, J. D. (2007). CPEB: a life in translation. Trends Biochem Sci 32, 279-285. Roberts, T. M., Pavalko, F. M., and Ward, S. (1986). Membrane and cytoplasmic proteins are transported in the same organele complex during nematode spermatogenesis. J Cel Biol 102, 1787-1796. 192 Roberts, T. M., and Ward, S. (1982). Centripetal flow of pseudopodial surface components could propel the amoeboid movement of Caenorhabditis elegans spermatozoa. J Cel Biol 92, 132-138. Ross, J. M., Kalis, A. K., Murphy, M. W., and Zarkower, D. (2005). The DM domain protein MAB-3 promotes sex-specific neurogenesis in C. elegans by regulating bHLH proteins. Dev Cel 8, 881-892. Roy, P. J., Stuart, J. M., Lund, J., and Kim, S. K. (2002). Chromosomal clustering of muscle-expresed genes in Caenorhabditis elegans. Nature 418, 975-979. Salvat, C., Acquaviva, C., Schefner, M., Robbins, I., Piechaczyk, M., and Jariel- Encontre, I. (2000). Molecular characterization of the thermosensitive E1 ubiquitin- activating enzyme cel mutant A31N-ts20. Requirements upon diferent levels of E1 for the ubiquitination/degradation of the various protein substrates in vivo. Eur J Biochem 267, 3712-3722. Sasagawa, Y., Yamanaka, K., and Ogura, T. (2007). ER E3 ubiquitin ligase HRD-1 and its specific partner chaperone BiP play important roles in ERAD and developmental growth in Caenorhabditis elegans. Genes Cels 12, 1063-1073. Savage, C., Das, P., Fineli, A. L., Townsend, S. R., Sun, C. Y., Baird, S. E., and Padget, R. W. (1996). Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc Natl Acad Sci U S A 93, 790-794. Sawada, H., Sakai, N., Abe, Y., Tanaka, E., Takahashi, Y., Fujino, J., Kodama, E., Takizawa, S., and Yokosawa, H. (2002). Extracelular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor. Proc Natl Acad Sci U S A 99, 1223-1228. Schaefer, H., and Rongo, C. (2006). KEL-8 is a substrate receptor for CUL3-dependent ubiquitin ligase that regulates synaptic glutamate receptor turnover. Mol Biol Cel 17, 1250-1260. Schefner, M., Nuber, U., and Huibregtse, J. M. (1995). Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 81-83. 193 Schefner, M., Smith, S., and Jentsch, S. (1998). The ubiquitin-conjugation system. (New York: Plenum Pres). Schnel, J. D., and Hicke, L. (2003). Non-traditional functions of ubiquitin and ubiquitin- binding proteins. J Biol Chem 278, 35857-35860. Schvarzstein, M., and Spence, A. M. (2006). The C. elegans sex-determining GLI protein TRA-1A is regulated by sex-specific proteolysis. Dev Cel 11, 733-740. Schwartz, D. C., and Hochstraser, M. (2003). A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem Sci 28, 321-328. Schwartz, H. T., and Horvitz, H. R. (2007). The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9. Genes Dev 21, 3181-3194. Segal, S. P., Graves, L. E., Verheyden, J., and Goodwin, E. B. (2001). RNA-Regulated TRA-1 nuclear export controls sexual fate. Dev Cel 1, 539-551. Setoyama, D., Yamashita, M., and Sagata, N. (2007). Mechanism of degradation of CPEB during Xenopus oocyte maturation. Proc Natl Acad Sci U S A 104, 18001- 18006. Seydoux, G., and Braun, R. E. (2006). Pathway to totipotency: lesons from germ cels. Cel 127, 891-904. Seydoux, G., and Dunn, M. A. (1997). Transcriptionaly represed germ cels lack a subpopulation of phosphorylated RNA polymerase I in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development 124, 2191- 2201. Seydoux, G., and Fire, A. (1994). Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans. Development 120, 2823-2834. Seydoux, G., Melo, C. C., Petit, J., Wood, W. B., Pries, J. R., and Fire, A. (1996). Represion of gene expresion in the embryonic germ lineage of C. elegans. Nature 382, 713-716. 194 Shakes, D. C., Sadler, P. L., Schumacher, J. M., Abdolrasulnia, M., and Golden, A. (2003). Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cel cycle abnormalities. Development 130, 1605- 1620. Shakes, D. C., and Ward, S. (1989a). Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev Biol 134, 189-200. Shakes, D. C., and Ward, S. (1989b). Mutations that disrupt the morphogenesis and localization of a sperm-specific organele in Caenorhabditis elegans. Dev Biol 134, 307-316. Shen, M. M., and Hodgkin, J. (1988). mab-3, a gene required for sex-specific yolk protein expresion and a male-specific lineage in C. elegans. Cel 54, 1019-1031. Shimada, M., Kanematsu, K., Tanaka, K., Yokosawa, H., and Kawahara, H. (2006). Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans. Mol Biol Cel 17, 5356-5371. Siepmann, T. J., Bohnsack, R. N., Tokgoz, Z., Baboshina, O. V., and Has, A. L. (2003). Protein interactions within the N-end rule ubiquitin ligation pathway. J Biol Chem 278, 9448-9457. Singson, A., Hil, K. L., and L'Hernault, S. W. (1999). Sperm competition in the absence of fertilization in Caenorhabditis elegans. Genetics 152, 201-208. Slack, F. J., Bason, M., Liu, Z., Ambros, V., Horvitz, H. R., and Ruvkun, G. (2000). The lin-41 RBC gene acts in the C. elegans heterochronic pathway betwen the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cel 5, 659-669. Smith, J. A., McGar, P., and Gileard, J. S. (2005). The Caenorhabditis elegans GATA factor elt-1 is esential for diferentiation and maintenance of hypodermal seam cels and for normal locomotion. J Cel Sci 118, 5709-5719. Snee, M., Benz, D., Jen, J., and Macdonald, P. M. (2008). Two distinct domains of Bruno bind specificaly to the oskar mRNA. RNA Biol 5. 195 Sonnevile, R., and Gonczy, P. (2004). Zyg-11 and cul-2 regulate progresion through meiosis I and polarity establishment in C. elegans. Development 131, 3527-3543. Sonnichsen, B., Koski, L. B., Walsh, A., Marschal, P., Neumann, B., Brehm, M., Aleaume, A. M., Artelt, J., Betencourt, P., Casin, E., et al. (2005). Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462- 469. Soustele, L., Roy, N., Ragone, G., and Giangrande, A. (2008). Control of gcm RNA stability is necesary for proper glial cel fate acquisition. Mol Cel Neurosci 37, 657-662. Standart, N., and Jackson, R. J. (2007). MicroRNAs repres translation of m7Gppp- capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation. Genes Dev 21, 1975-1982. Starostina, N. G., Lim, J. M., Schvarzstein, M., Wels, L., Spence, A. M., and Kipreos, E. T. (2007). A CUL-2 ubiquitin ligase containing thre FEM proteins degrades TRA- 1 to regulate C. elegans sex determination. Dev Cel 13, 127-139. Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1996). The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cel cycle-dependent manner. J Biol Chem 271, 15608-15614. Stephen, A. G., Trausch-Azar, J. S., Handley-Gearhart, P. M., Ciechanover, A., and Schwartz, A. L. (1997). Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation. J Biol Chem 272, 10895-10903. Subramanian, L., Benson, M. D., and Iniguez-Lluhi, J. A. (2003). A synergy control motif within the atenuator domain of CAT/enhancer-binding protein alpha inhibits transcriptional synergy through its PIASy-enhanced modification by SUMO-1 or SUMO-3. J Biol Chem 278, 9134-9141. Sulston, J. E., and Horvitz, H. R. (1977). Post-embryonic cel lineages of the nematode, Caenorhabditis elegans. Dev Biol 56, 110-156. 196 Sulston, J. E., Schierenberg, E., White, J. G., and Thomson, J. N. (1983). The embryonic cel lineage of the nematode Caenorhabditis elegans. Dev Biol 100, 64-119. Sung, P., Berleth, E., Pickart, C., Prakash, S., and Prakash, L. (1991). Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme. Embo J 10, 2187-2193. Surdo, P. L., Bottomley, M. J., Satler, M., and Schefzek, K. (2003). Crystal structure and nuclear magnetic resonance analyses of the SAND domain from glucocorticoid modulatory element binding protein-1 reveals deoxyribonucleic acid and zinc binding regions. Mol Endocrinol 17, 1283-1295. Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Thompson, W. E., and Schaten, G. (2001). A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mamalian epididymis. J Cel Sci 114, 1665-1675. Suzuki, Y., Yandel, M. D., Roy, P. J., Krishna, S., Savage-Dunn, C., Ross, R. M., Padget, R. W., and Wood, W. B. (1999). A BMP homolog acts as a dose- dependent regulator of body size and male tail paterning in Caenorhabditis elegans. Development 126, 241-250. Swanson, R., and Hochstraser, M. (2000). A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in Sacharomyces cerevisiae. FEBS Let 477, 193-198. Tenenhaus, C., Schubert, C., and Seydoux, G. (1998). Genetic requirements for PIE-1 localization and inhibition of gene expresion in the embryonic germ lineage of Caenorhabditis elegans. Dev Biol 200, 212-224. Teng, F. Y., and Tang, B. L. (2005). APC/C regulation of axonal growth and synaptic functions in postmitotic neurons: the Liprin-alpha connection. Cel Mol Life Sci 62, 1571-1578. Tenlen, J. R., Schisa, J. A., Diede, S. J., and Page, B. D. (2006). Reduced dosage of pos-1 suppreses Mex mutants and reveals complex interactions among CCH zinc-finger proteins during Caenorhabditis elegans embryogenesis. Genetics 174, 1933-1945. 197 Thrower, J. S., Hoffman, L., Rechsteiner, M., and Pickart, C. M. (2000). Recognition of the polyubiquitin proteolytic signal. Embo J 19, 94-102. Timons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. Trausch, J. S., Grenfel, S. J., Handley-Gearhart, P. M., Ciechanover, A., and Schwartz, A. L. (1993). Imunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton. Am J Physiol 264, C93-102. Varkey, J. P., Muhlrad, P. J., Minniti, A. N., Do, B., and Ward, S. (1995). The Caenorhabditis elegans spe-26 gene is necesary to form spermatids and encodes a protein similar to the actin-asociated proteins kelch and scruin. Genes Dev 9, 1074-1086. Vermeirsen, V., Barasa, M. I., Hidalgo, C. A., Babon, J. A., Sequera, R., Doucete- Stam, L., Barabasi, A. L., and Walhout, A. J. (2007). Transcription factor modularity in a gene-centered C. elegans core neuronal protein-DNA interaction network. Genome Res 17, 1061-1071. Walden, H., Podgorski, M. S., and Schulman, B. A. (2003). Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NED8. Nature 422, 330-334. Walker, A. K., Boag, P. R., and Blackwel, T. K. (2007). Transcription reactivation steps stimulated by oocyte maturation in C. elegans. Dev Biol 304, 382-393. Ward, S., Argon, Y., and Nelson, G. A. (1981). Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans. J Cel Biol 91, 26-44. Ward, S., and Carel, J. S. (1979). Fertilization and sperm competition in the nematode Caenorhabditis elegans. Dev Biol 73, 304-321. Ward, S., Hogan, E., and Nelson, G. A. (1983). The initiation of spermiogenesis in the nematode Caenorhabditis elegans. Dev Biol 98, 70-79. Ward, S., and Klas, M. (1982). The location of the major protein in Caenorhabditis elegans sperm and spermatocytes. Dev Biol 92, 203-208. 198 Ward, S., Roberts, T. M., Strome, S., Pavalko, F. M., and Hogan, E. (1986). Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins. J Cel Biol 102, 1778-1786. Weisman, A. M. (2001). Themes and variations on ubiquitylation. Nat Rev Mol Cel Biol 2, 169-178. Wiborg, O., Pedersen, M. S., Wind, A., Berglund, L. E., Marcker, K. A., and Vuust, J. (1985). The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. Embo J 4, 755-759. Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H., and Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat Genet 28, 160-164. Wiliamson, A., and Lehmann, R. (1996). Germ cel development in Drosophila. Annu Rev Cel Dev Biol 12, 365-391. Wojciak, J. M., and Clubb, R. T. (2001). Finding the function buried in SAND. Nat Struct Biol 8, 568-570. Wolf, N., Hirsh, D., and McIntosh, J. R. (1978). Spermatogenesis in males of the fre- living nematode, Caenorhabditis elegans. J Ultrastruct Res 63, 155-169. Xu, L., Wei, Y., Reboul, J., Vaglio, P., Shin, T. H., Vidal, M., Eledge, S. J., and Harper, J. . (2003). BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425, 316-321. Xu, X. Z., and Sternberg, P. W. (2003). A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization. Cel 114, 285-297. Yanai, I., Baugh, L. R., Smith, J. J., Roehrig, C., Shen-Or, S. S., Clagget, J. M., Hil, A. A., Slonim, D. K., and Hunter, C. P. (2008). Pairing of competitive and topologicaly distinct regulatory modules enhances paterned gene expresion. Mol Syst Biol 4, 163. Yang, F., Vought, B. W., Saterle, J. S., Walker, A. K., Jim Sun, Z. Y., Wats, J. L., DeBeaumont, R., Saito, R. M., Hyberts, S. G., Yang, S., et al. (2006). An 199 ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis. Nature 442, 700-704. Yi, W., Ross, J. M., and Zarkower, D. (2000). Mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior. Development 127, 4469-4480. Yokota, N., and Sawada, H. (2007). Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the viteline envelope during fertilization of the sea urchin Pseudocentrotus depresus. Dev Biol 308, 222-231. Zacksenhaus, E., Sheinin, R., and Wang, H. S. (1990). Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cel defect in DNA replication and cel cycle progresion to Xp11.2--p11.4. Cytogenet Cel Genet 53, 20-22. Zarkower, D. Somatic sex determination, In WormBook, T. C. e. R. Community, ed. (WormBook). Zarkower, D., and Hodgkin, J. (1992). Molecular analysis of the C. elegans sex- determining gene tra-1: a gene encoding two zinc finger proteins. Cel 70, 237-249. Zhang, F., Barboric, M., Blackwel, T. K., and Peterlin, B. M. (2003). A model of represion: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb. Genes Dev 17, 748-758. Zhao, C., Beaudenon, S. L., Keley, M. L., Waddel, M. B., Yuan, W., Schulman, B. A., Huibregtse, J. M., and Krug, R. . (2004). The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein. Proc Natl Acad Sci U S A 101, 7578-7582. Zhu, G. D., and L'Hernault, S. W. (2003). The Caenorhabditis elegans spe-39 gene is required for intracelular membrane reorganization during spermatogenesis. Genetics 165, 145-157. Zhu, H., Zhou, Z. M., Huo, R., Huang, X. Y., Lu, L., Lin, M., Wang, L. R., Zhou, Y. D., Li, J. M., and Sha, J. H. (2004). Identification and characteristics of a novel E1 like gene nUBE1L in human testis. Acta Biochim Biophys Sin (Shanghai) 36, 227-234.