ABSTRACT Title of dissertation: RNA SILENCING AND HIGHER ORDER CHROMATIN ORGANIZATION IN DROSOPHILA Nellie Moshkovich, Doctor of Philosophy, 2011 Dissertation directed by: Dr. Elissa P. Lei NIH, NIDDK, LCDB David A. O?Brochta University of Maryland, Department of Entomology Higher order chromatin organization influences gene expression, but mechanisms by which this phenomenon occurs are not well understood. RNA silencing, a conserved mechanism that involves small RNAs bound to an Argonaute protein, mediates gene expression via transcriptional or post-transcriptional regulation. Recently, a role for RNA silencing in chromatin has been emerging. In fission yeast, a major role of RNA interference (RNAi) is to establish pericentromeric heterochromatin. However, whether this mechanism is conserved throughout evolution is unclear. In Drosophila, a powerful model organism, there are multiple functionally distinct RNA silencing pathways. Previous studies have suggested the involvement of the Piwi-interacting RNA (piRNA) and endogenous small interfering RNA (endo-siRNA) pathways in heterochromatin formation in order to silence transposable elements in germline and somatic tissues, respectively, but direct evidence is lacking. We addressed whether the genomic locations generating these small RNAs may act as AGO-dependent platforms for heterochromatin recruitment. Our genetic and biochemical analyses revealed that heterochromatin is nucleated independently of endo-siRNA and piRNA pathways suggesting that RNAi- dependent heterochromatin assembly may not be conserved in metazoans. Chromatin insulators are regulatory elements characterized by enhancer blocking and barrier activity. Insulators form large nuclear foci termed insulator bodies that are tethered to the nuclear matrix and have been proposed to organize the genome into distinct transcriptional domains by looping out intervening DNA. In Drosophila, RNA silencing has been reported to affect nuclear organization of gypsy insulator complexes and formation of Polycomb repression bodies. Our studies revealed that AGO2 is required for CTCF/CP190-dependent Fab-8 insulator function independent of its catalytic activity or Dicer-2. Moreover, AGO2 associates with euchromatin but not heterochromatin genome-wide. Also, AGO2 associates physically with CP190 and CTCF, and mutation of CTCF, CP190, or AGO2 decreases chromosomal looping interactions and alters gene expression. We propose a novel RNAi-independent role for AGO2 in the nucleus. We postulate that insulator proteins recruit AGO2 to chromatin to promote or stabilize chromosomal interactions crucial for proper gene expression. Overall, our findings demonstrate novel mechanisms by which RNA silencing affects gene expression on the level of higher order chromatin organization. RNA SILENCING AND HIGHER ORDER CHROMATIN ORGANIZATION IN DROSOPHILA by Nellie Moshkovich Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2011 Advisory Committee: Professor David A. O?Brochta, Chair Dr. Elissa P. Lei, Co-chair Professor Leslie Pick Professor Stephen M. Mount Professor Jonathan D. Dinman Professor Judd O. Nelson ?Copyright by Nellie Moshkovich 2011 DEDICATION I would like to dedicate this work to my family; to my mother, Galina, for teaching me the value of hard work, determination and perseverance, and for instilling in me the inspiration to set high goals and the confidence to achieve them; to my father, Yuriy, for emphasizing to me the importance of science and encouraging me in my academic pursuits; to my husband, Igor, my emotional anchor, for his cool head and warm heart; and to my daughter, Nikita, my little bookworm, so that she may be proud of me as much as I am proud of her. ii ACKNOWLEDGEMENTS My deepest gratitude goes to my advisor, Elissa P. Lei. I have been fortunate to have an advisor who not only provided me with rigorous scientific training but also cared for all my successes and failures as if they were her own. Thank you for sharing your enthusiasm and dedication to science with me, it was really inspiring. Thank you also for leading by example, holding me to a high research standard, and for taking a chance on a rather unconventional graduate student. I would also like to acknowledge the members of my dissertation committee, David A. O?Brochta, Leslie Pick, Stephen M. Mount and Jonathan D. Dinman for their guidance, encouragement and constructive criticism throughout the years. I would like to thank the members of Lei lab, past and present, for their indispensable assistance in my research and writing efforts over the last four years. I would like in particular to acknowledge the colleagues I collaborated with - Patrick Boyle, for his many empirical and intellectual contributions, the many valuable discussions, and his friendship; Parul Nisha, for laying the foundation for some of my research, her support and invaluable advice on graduate school and science; Ryan Dale, for unlimited computational support and sharing the magic of computational biology with me; Leah Matzat, for insightful discussions, reading of numerous drafts, her infectious enthusiasm and camaraderie. I would also like to thank Brandi Thompson, Matthew Emmett, Patrick Murphy and Madoka Chinen. I extend my appreciation to Erik Baehrecke for jump starting my graduate career and for teaching me the basics of fly husbandry. Many thanks go to my friends and colleagues from Baehrecke lab, who made the first two years of graduate iii school more bearable, Christina Kary McPhee, Michelle Beaucher, Deb Berry, Jahda Hill, Yakup Batlevi and Sudeshna Dutta. Finally, this journey would not have been possible if it were not for my parents. It is through their personal sacrifice that I had the luxury to strive for academic excellence and I am indebted to them. I also would like to thank my husband for keeping everything in perspective for me and for his love and care. Last but not least, I acknowledge my daughter who has grown into a wonderful human being while her mom was occasionally absent chasing laurels. iv TABLE OF CONTENTS LIST OF TABLES ????????????????????????.. viii LIST OF FIGURES ???????????????????????... ix LIST OF ABBREVIATIONS ???????????????????? xi CHAPTER 1. Introduction ????????????????????... 1 RNA silencing ??????????????????????????... 1 Transposable element silencing by the piRNA and endo-siRNA pathways ?? 3 A role for RNA silencing in the nucleus ???????????????.. 7 Heterochromatin and RNA silencing ?????????????????? 8 RNAi-mediated heterochromatic silencing in S. pombe ?????????.. 9 RNA silencing involvement in heterochromatin recruitment in metazoans ?? 11 Polycomb-mediated gene repression and RNA silencing ??????????. 13 Chromatin insulators, nuclear organization and RNA silencing ???????... 14 Conclusion ???????????????????????????? 20 CHAPTER 2. HP1 recruitment in the absence of Argonaute proteins in Drosophila ???????????????????????????? 23 Abstract ?????????????????????????????. 23 Introduction ??????????????????????????...? 25 Results ?????????????????????????????... 29 Heterochromatin dependent transcriptional silencing at piRNA clusters ??? 29 piRNA and endo-siRNA pathway mutants decrease transcription at piRNA clusters ????????????????????????????. 37 HP1 chromatin association is increased at piRNA clusters in somatic tissues of RNA silencing mutants ?????????????????????.. 40 HP1 also associates with piRNA clusters in ovaries ??????????.. 48 HP1 chromatin association is not affected greatly by depletion of Piwi in somatic ovarian follicle cells ???????????????????? 50 Loss of piRNA production from a single cluster results in global HP1 mislocalization ?????????????????????????. 53 Mutation of the flam piRNA cluster suppresses heterochromatic silencing at a distant site ??????????????????????????? 58 Discussion ????????????????????????????. 59 v AGO2 and Piwi are not required for HP1 association at piRNA clusters ??... 59 Additional candidate platforms for Piwi-dependent HP1 recruitment ???? 63 Functions for piwi outside of the gonad ???????????????.. 63 HP1 redistribution in piRNA pathway mutants ????????????.. 64 Conclusions ??????????????????????????. 65 Acknowledgements ????????????????????????.. 67 Author contributions ????????????????????????. 68 Materials and Methods ???????????????????????. 69 CHAPTER 3. RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function ???????????????????? 77 Abstract ?????????????????????????????. 77 Introduction ???????????????????????????... 78 Results ?????????????????????????????.. 82 AGO2 associates with euchromatin and not repetitive sequences ?????.. 82 AGO2 colocalizes with chromatin insulator sites throughout the genome ??.. 94 AGO2 associates with active promoters ???????????????.. 95 AGO2 chromatin association does not correspond to regions of the genome that produce endo-siRNA ??????????????????????? 96 AGO2 binds to PREs and overlaps extensively with TrxG and PcG proteins ? 100 AGO2 opposes Polycomb function ?????????????????. 104 AGO2 but not its catalytic activity is specifically required for Fab-8 insulator activity ????????????????????????????. 109 AGO2 interacts physically with CTCF and CP190 ???????????. 114 AGO2 chromatin association requires CP190 and CTCF ????????... 118 AGO2 associates with chromatin downstream of CTCF and CP190 ????. 122 CP190, CTCF and AGO2 are required for looping at the Abd-B locus ???.. 125 AGO2 is required for proper expression of Abd-B similar to CTCF ????... 126 Discussion ????????????????????????????. 127 AGO2 localizes predominantly to euchromatin and not heterochromatin ??.. 127 RNAi-independent function for AGO2 at chromatin ??????????.. 128 Role of AGO2 in Fab-8 insulator function ??????????????.. 128 Role of AGO2 in long range chromosomal interactions ?????????.. 130 Conclusions ??????????????????????????.. 131 Acknowledgements ????????????????????????.. 134 Author contributions ????????????????????????. 135 Materials and Methods ............................................................................................. 136 vi CHAPTER 4. Discussion and future directions ????????????. 154 RNA silencing and its role in heterochromatin assembly ?????????? 154 piRNA- and endo-siRNA-mediated TE silencing ???????????... 154 Alternative mechanisms for heterochromatin nucleation ????????? 156 RNA silencing and its effects on chromatin insulators ???????????. 157 AGO2: a multifunctional protein ??????????????????. 157 Role for AGO2 in transcriptional regulation ?????????????... 159 A connection between two functions of AGO2 ????????????... 161 AGO2 role in nuclear organization ?????????????????.. 163 Conclusion????????????????????????????.. 164 REFERENCES ?????????????????????????? 166 vii LIST OF TABLES Table 2-1. Expression of mini-white in fly lines harboring P element insertions in four top piRNA clusters ??????????????????????? 32 Table 2-2. Primer set sequences used for ChIP at the flam piRNA cluster ???. 75 Table 2-3. Primer set sequences used for ChIP at the 80EF piRNA cluster ?...? 76 Table 3-1. Sources of data for tiling arrays, endo-siRNA, and genome features ? 150 Table 3-2. List of primers ?????????????????????? 152 viii LIST OF FIGURES Figure 1-1. Drosophila piRNA and siRNA pathways. ??????????... 6 Figure 1-2. Model for RNAi-mediated centromeric silencing in S. pombe. ??.. 10 Figure 1-3. Functional properties of chromatin insulators. ????????? 15 Figure 1-4. Nuclear organization of a chromatin insulator. ????????? 17 Figure 1-5. Abd-B cis-regulatory region of the bithorax complex (BX-C). ??? 19 Figure 2-1. Schematic representation of four top piRNA clusters. ??????. 31 Figure 2-2. piRNA and endo-siRNA pathway mutants display increased silencing of transcriptional reporters at or near piRNA clusters. ???????????.. 35 Figure 2-3. Dcr-2 mutants display increased HP1 chromatin association and increased silencing at piRNA clusters. ?????????????????. 38 Figure 2-4. HP1 associates with chromatin at piRNA clusters, and its levels increase in RNA silencing mutants. ????????????????..?? 42 Figure 2-5. Su(Hw) does not associate with chromatin at piRNA clusters in heads. ??????????????????????????????. 43 Figure 2-6. HP1 chromatin association levels are increased in piwi mutants at piRNA clusters. ?????????????????????????.? 44 Figure 2-7. HP1 chromatin association levels are increased in AGO2 mutants at piRNA clusters. ????????????????????????.?? 45 Figure 2-8. HP1 protein levels in wildtype, flam1, AGO251B and piwi1/piwi2 fly heads. ????????????????????????????..?.. 46 Figure 2-9. HP1 associates with chromatin at piRNA clusters in ovaries. ??.? 48 Figure 2-10. Depletion of Piwi from ovarian somatic follicle cells does not affect HP1 recruitment to piRNA clusters. ?????????????????.? 51 Figure 2-11. Mutation of the flam piRNA cluster results in global HP1 redistribution. ??????????????????????????? 55 Figure 2-12. spn-Ehls-E1/spn-Ehls-E616 mutants display accumulation of HP1 at the chromocenter. ??????????????????????????? 56 ix Figure 2-13. ChIP primer efficiency and specificity. ??????????..? 74 Figure 3-1. ChIP-seq profiles of AGO2 in S2 and S3 cells at BX-C. ?????. 85 Figure 3-2. Overlap between genome-wide binding sites of AGO2, insulator, TrxG/PcG, transcription related factors, and promoters. ??????????.. 92 Figure 3-3. Distribution of endo-siRNA densitities for AGO2 binding sites compared to 3? cis-NATs and transcriptionally active or inactive regions. ???.. 99 Figure 3-4. AGO2 behaves as a TrxG protein. ?????????????... 103 Figure 3-5. Effects of Pc and TRX knock-down on AGO2 association with chromatin. ???????????????????????????..? 108 Figure 3-6. AGO2 but not its catalytic activity is required for Fab-8 insulator function. ????????????????????????????? 113 Figure 3-7. AGO2 associates physically with CP190. ??????????.. 117 Figure 3-8. AGO2 is required for looping interactions throughout the Abd-B locus and proper gene expression. ???????????????????. 124 Figure 3-9. Model for AGO2 function with respect to CTCF/CP190 chromatin insulator activity. ?????????????????????????... 133 x LIST OF ABBREVIATIONS Abd-B Abdominal-B AGO Argonaute Aub Aubergine BX-C Bithorax complex CP190 Centrosomal protein 190 3C Chromosome conformation capture ChIP Chromatin immunoprecipitation CTCF CCCTC-binding factor Dcr Dicer dsRNA Double stranded RNA endo-siRNA Endogenous siRNA flam flamenco Fab-8 Frontabdominal-8 GAF GAGA factor HP1 Heterochromatin protein 1 IP Immunoprecipitation KD Knock down Mod(mdg4)2.2 Modifier of mdg4 2.2 NAT Natural antisense transcript OSC Ovarian somatic cell piRNA Piwi-interacting RNA PEV Position-effect variegation PcG Polycomb group PRE Polycomb response element RNAi RNA interference RITS RNA-induced transcriptional silencing complex siRNA Short interfering RNA Su(Hw) Suppressor of Hairy wing Su(var) Suppressor of variegation TSS Transcription start site TE Transposable element TrxG Trithorax group WT wildtype xi CHAPTER 1 INTRODUCTION Eukaryotic genomes are organized into a complex system where physical interactions between genes and regulatory elements ensure proper regulation of gene expression. It has become apparent that nuclear organization and chromatin folding are vital for spatial and temporal control necessary for proper gene expression during development and differentiation. The mechanisms regulating nuclear organization still need to be addressed. Gaining mechanistic insight into the function of key regulators mediating higher order chromatin organization as well as elucidating new contributors may aid in understanding abnormal biological processes such as tumorigenesis in which gene expression is altered. Recently, RNA silencing has been emerging as one such contributing factor. RNA SILENCING RNA silencing pathways are evolutionarily conserved mechanisms that control gene expression via sequence-specific interactions mediated by a small RNA bound to an Argonaute (AGO) effector protein and are involved in numerous biological processes such as post-transcriptional gene regulation, defense against transposable elements (TEs) and pathogens, and chromatin organization. RNA silencing pathways are characterized by the activity of an Argonaute effector protein that binds small RNAs directly. Ranging 1 from one AGO gene in Schizosaccharomyces pombe ( S. pombe) to 27 in Caenorhabditis elegans (C. elegans), the number varies greatly among species. The five Argonautes in Drosophila melanogaster can be divided into two families based on homology. The AGO subfamily includes AGO1 and AGO2, and the Piwi subfamily consists of Piwi, Aubergine (Aub), and AGO3 (reviewed in Hutvagner and Simard 2008). Drosophila AGO1 and AGO2 are expressed ubiquitously (Williams and Rubin 2002; Rehwinkel et al. 2006). AGO1 is primarily required for the microRNA pathway, which regulates mRNA expression and functions chiefly through mRNA destabilization as well as translational repression (reviewed in Czech and Hannon 2011). Existing as a mechanism to protect against exogenous double stranded RNA (dsRNA), AGO2 associates with 21- 22 nt short interfering RNA (siRNA) produced by Dicer-2 (Dcr-2) in a pathway that is required for viral immunity and a robust RNAi response (Hammond et al. 2001; Wang and Ligoxygakis 2006). In addition, AGO2 binds endogenous siRNAs (endo-siRNAs) that mediate TE silencing in somatic tissues (Chung et al. 2008; Czech et al. 2008; Ghildiyal et al. 2008; Kawamura et al. 2008). The Piwi clade of AGO proteins mediates TE silencing in the gonad. The expression of piwi, aub, and AGO3 is mainly, although not exclusively, in the gonad (Williams and Rubin 2002; Saito et al. 2006; Br ennecke et al. 2007; Brower-Toland et al. 2007). Whereas Piwi is a nuclear protein that is detected in both germ and somatic cells in Drosophila ovaries, Aub and AGO3 localize to the cytoplasm and accumulate in the nuage, an electron-dense perinuclear structure associated with germline RNA processing (Cox et al. 2000; Saito et al. 2006; Brenn ecke et al. 2007; Gunawardane et al. 2007; Nishida et al. 2007). The Piwi proteins asso ciate with a class of small RNAs termed 2 Piwi-interacting RNAs (piRNAs) involved in the repression of TEs in the germline (Saito et al. 2006; Brennecke et al. 2007; Ni shida et al. 2007; Yin and Lin 2007). All AGO proteins are characterized by three functional domains crucial for their interaction with small RNAs: the PAZ, Mid and PIWI domains. The PAZ and Mid domains bind small RNAs (Song et al. 2003; Ma et al. 2004). The PIWI domain is a catalytically active RNase H-like domain that cleaves the targeted RNA molecule (Song et al. 2004; Ma et al. 2005). The endonuclease activity of AGO proteins is often referred to as Slicer activity. Slicer activity of certain Drosophila AGO proteins has been shown to be vital for biological functions such as small RNA-based TE and pathogen defense. The role of AGO proteins in post-transcriptional gene silencing is rather well- characterized. Other aspects of AGO function such as its roles in transcription and chromatin organization or yet unknown functions will be elucidated in the future. Transposable element silencing by the piRNA and endo-siRNA pathways Eukaryotic genomes are beset with TEs, mobile genetic elements, transposition and recombination of which can cause genetic instability leading to deleterious mutations. Suppression of TEs is especially imperative in the gonad in order to limit the propagation of unwanted mutations and is achieved principally by the activity of the Piwi subfamily of AGO proteins. In Drosophila, Piwi, Aub, and AGO3 bind to 23-30 nt piRNAs that are predominantly derived from genomic locations termed piRNA clusters (Brennecke et al. 2007; Yin and Lin 2007). These piRNA producing loci are mainly pericentromeric and enriched in transposon sequences. Based on comparative sequence 3 analysis of piRNAs immunopurified from the ovary, the ?ping-pong? or ?amplification loop? model for germline piRNA biogenesis wa s proposed (Figure1-1; Brennecke et al. 2007; Gunawardane et al. 2007). Precursor tr anscripts from piRNA clusters, derived from either one or both strands, give rise to piRNAs bound by Piwi, Aub, or AGO3. Those piRNAs antisense to a homologous TE transcript can result in its cleavage, and this event defines the 5? end of a secondary piRNA that can then bind and cleave an antisense piRNA cluster transcript, and the cycle can continue. Piwi appears to play a minor role in ping-pong piRNA amplification (Li et al. 2009a; Malone et al. 2009), which is thought to occur primarily in the cytoplasmic nuage driven by Aub and AGO3 (Harris and Macdonald 2001; Brennecke et al. 2007; Gunawardane et al. 2007). Piwi independently serves an additional role in the silencing of certain TEs expressed in somatic follicle cells surrounding the ovary. This somatic piRNA pathway depends on Piwi alone and therefore does not undergo ping-pong amplification (Li et al. 2009a; Malone et al. 2009; Saito et al. 2009). The flamenco (flam) piRNA cluster, which controls the gypsy, ZAM , and Idefix retrotransposons (Prud'homme et al. 1995; Desset et al. 2003), is one of the major sites of primary piRNA production (Lau et al. 2009; Li et al. 2009a; Malone et al. 2009; Saito et al. 2009). Piwi associates with piRNAs generated by flam and other piRNA clusters, and has been proposed to cleave homologous TE transcripts using its Slicer activity (Saito et al. 2006). TE silencing in somatic tissues is mediated by AGO2. AGO2 binds endo- siRNAs, the majority of which silence the expression of TEs outside of the gonad (Chung et al. 2008; Czech et al. 2008; Gh ildiyal et al. 2008; Kawamura et al. 2008). Silencing is achieved by Dcr-2-mediated cleavage of dsRNAs into 21-22 nt siRNA that are loaded 4 into AGO2, which cleaves the target TE mRNA using its Slicer activity. Interestingly, many endo-siRNAs map to the same genomic regions that generate piRNAs. Although some redundancy in TE targeting between the two pathways is plausible, some TEs were shown to be silenced by only one of these pathways (Watanabe et al. 2008). 5 Figure 1-1. Drosophila piRNA and siRNA pathways. A. piRNA pathway. Single- stranded piRNA precursors are generated from either uni-strand piRNA clusters that are transcribed in one direction or dual-strand clusters that produce piRNAs from both strands. After primary processing, piRNAs derived from uni-strand clusters are loaded into Piwi to target TEs in ovarian somatic cells. Aub and AGO3-loaded piRNAs originating from the dual-strand clusters are further amplified in the ?ping-pong? cycle targeting TEs in ovarian germline cells. B. Endo-siRNA pathway. Most endo-siRNAs are derived from TE transcripts. DsRNA precursors are processed by Dcr-2 and Loqs. Endo-siRNAs are loaded onto AGO2. AGO2-endo-siRNA complexes target TE transcripts cleaving them by AGO2 Slicer activity. Modified from Siomi et al., Nat Rev Gen 2011. TE silencing Piwi Piwi TE silencing TE silencing Transcription Primary processing AGO3 AGO3 Aub/ Piwi Ping pong cycle Aub/ Piwi Loading TE transcript dual-strand piRNA cluster transcript A uni-strand dual-strand B piRNA cluster piRNA cluster TE Transcription Dcr-2 Loq s dsRNA AGO2 AGO2 AGO2 AGO2 Slicing Loading endo-siRNA 6 A role for RNA silencing in the nucleus Although RNA silencing was originally believed to be a cytoplasmic mechanism, there is a sufficient amount of empirical evidence indicating that it can also function in the nucleus in various organisms. In S. pombe, RNAi functions primarily to nucleate and maintain heterochromatin required for centromere function but also has a function in euchromatin. In the unicellular ciliate Tetrahymena thermophilica (T. thermophilica ), the RNAi machinery triggers programmed genome elimination that protects against potentially harmful TEs, which are also silenced by heterochromatin recruitment (reviewed in Mochizuki 2010). In C. elegans, NRDE-3, an AGO protein, can localize to the nucleus upon siRNA loading (Guang et al. 2008). NRDE-3 along with NRDE-2 has also been shown to inhibit RNA Polymerase II (Pol II) elongation (Guang et al. 2010). In addition, another AGO protein, CSR-1, mediates a pathway that affects meiotic silencing of unpaired chromatin and chromosome segregation during early embryogenesis (Claycomb et al. 2009; van Wolfswinkel et al. 2009). Recently, a study reported human Ago1 and Ago2 localization to the nucleus in HeLa cells (Weinmann et al. 2009). Interestingly, in mammals, AGO1 and AGO2 guide synthetic siRNAs to gene promoters (Janowski et al. 2006; Kim et al. 2006). However, the specific mechanisms of AGO- directed RNA silencing in the nucleus and its function on chromatin remains vague in metazoans. 7 HETEROCHROMATIN AND RNA SILENCING Given the vast size and complexity of eukaryotic genomes, the genetic material must be efficiently packaged into the nucleus. The basic structural unit is a nucleosome, which consists of an octamer of core histones (H2A, H2B, H3, and H4) with 147 bp of DNA wrapped around it (Kornberg 1974). The individual nucleosomes are further arranged into 11 nm ?beads on a string? arrays that make up higher order structure known as chromatin. Microscopically, chromatin can be classified into two distinct forms, euchromatin and heterochromatin (reviewed in Richards and Elgin 2002). During interphase, euchromatin is visualized as decondensed chromatin whereas heterochromatin is the compacted form. In Drosophila, an estimated one-third of the genome is composed of repetitive and noncoding sequences associated with heterochromatin. This form of chromatin is characterized by repeat-rich sequences, hypoacetylation of histone tails, and dimethylation of histone H3 on lysine 9 (H3K9me2) (reviewed in (Grewal and Elgin 2007)). A conserved nonhistone Heterochro matin Protein 1 (HP1) is a critical component of heterochromatin, localizing predominantly at and near centromeres but also residing at telomeres, the Y, and fourth chromosomes. These regions tend to be rich in TEs, which must be suppressed in order to maintain genomic stability but can serve a cellular function, particularly in the case of Het-A and TART at the telomeres (reviewed in (Mason et al. 2008)). 8 RNAi-mediated heterochromatic silencing in S. pombe The paradigm for how RNA silencing controls gene expression at the chromatin level comes from pioneering genetic and biochemical studies in S. pombe, which have shed considerable light on mechanisms of heterochromatin assembly. The RNA interference (RNAi) machinery was found to play a key role in heterochromatin formation by detecting the transcription of specific DNA repeats located at the mating type locus and the centromere and subsequently nucleating heterochromatin. For example, double-stranded RNAs (dsRNA) transcribed from pericentromeric repeats are processed by the RNase III endonuclease Dicer1 into siRNAs (Figure1-2; Volpe et al. 2002). The single Argonaute, Argonaute1 binds these siRNAs as part of the RNA- induced transcriptional silencing complex (RITS) (Motamedi et al. 2004). Loading of RITS with siRNA and recruitment of the complex to the site of dsRNA transcription requires the Clr4 histone methyltransferase, which methylates H3K9 (Noma et al. 2004). This methylation mark serves as a binding site for Swi6, a fission yeast homolog of HP1, leading to heterochromatin establishment and spreading. Importantly, heterochromatin can also be nucleated independently of RNAi by other mechanisms. In fission yeast, for example, in the absence of RNAi the ATF/CREB stress-activated proteins promote heterochromatin formation at the mating type locus (Jia et al. 2004), and the Taz1 protein can establish HP1 recruitment to telomeres (Kanoh et al. 2005). These studies exemplify the redunda ncy of RNAi and additional mechanisms with respect to the formation of heterochromatin. 9 Figure 1-2. Model for RNAi-mediated centromeric silencing in S. pombe. In this pathway, dsRNAs transcribed from pericentromeric repeat-rich regions are processed by Dcr-1 into siRNAs that are loaded into the RITS complex containing Ago1. Association of the RITS complex with the pericentric repeats via sequence-specific pairing guides H3K9 methylation leading to heterochromatin nucleation and spreading. Modified from Verdel et al., Science, 2004. AGO1 AGO1 Dcr-1 AGO1 dsRNAs siRNAs Sequence-specific targeting Association with H3K9me Heterochromatin spreading Inactive RITS HP1 ? H3K9me2 ? 10 RNA silencing involvement in heterochromatin recruitment in metazoans Based on the model in S. pombe, it has been hypothesized that the mechanism of RNAi-dependent heterochromatin assembly is evolutionarily conserved between unicellular eukaryotes and metazoans. Multiple studies suggested that one or more Drosophila RNA silencing pathways may participate in transcriptional TE silencing by inducing heterochromatin formation, but direct evidence is lacking. Initial evidence that implicated piRNA pathways, and Piwi in particular, in establishment or maintenance of heterochromatin in the germline came from an observation that mutation of piwi, aub, or spn-E, encoding an RNA helicase required for the germline piRNA pathway (Vagin et al. 2006; Malone et al. 2009), resu lts in defects in heterochromatic silencing and visible changes in heterochromatin localization. These mutants display reduced silencing of pericentromeric transcriptional reporters and exhibit mislocalization of HP1 and H3K9me2 in salivary gland polytene chromosomes (Pal-Bhadra et al. 2004) suggesting a role for the piRNA silencing factors in nucleating or maintaining heterochromatin. These results, however, contrast with a study that investigated the role of Piwi on 3R-TAS subtelomeric region, which is a site of Piwi chromatin association and piRNA production. The study demonstrated that a mutation of piwi results in an increase of transcriptional silencing at 3R-TAS region and an increase of HP1 association suggesting an activating role for Piwi on chromatin (Yin and Lin 2007). Moreover, HP1 was identified as an interactor of Piwi in yeast two-hybrid screens (Brower-Toland et al. 2007). Furthermore, both proteins associate specifically with the chromatin of two transposable elements, 1360 and the F element . Based on their findings, the authors propose that Piwi could 11 serve as a recruitment platform for HP1 binding and heterochromatin-mediated silencing. However, this model appears not to be applicable to the 3R-TAS subtelomeric region. Thus, it remains an open question whether other sites in the genome could serve as Piwi- dependent HP1 recruitment sites. Two studies suggest that the endo-siRNA silencing pathway may participate in transcriptional TE silencing by inducing heterochromatin formation. First, mutation of AGO2 results in pleiotropic cellular defects in early embryos including mislocalization of HP1 and the histone H3 variant CID, which binds specifically the centromere (Deshpande et al. 2005). Later in development, AGO2 mutants display mislocalization of HP1 on polytene chromosomes of the larval salivary gland (Fagegaltier et al. 2009). Additionally, silencing of a pericentromeric transcriptional reporter is relieved when the maternally derived pool of AGO2 is reduced. Despite these defects, AGO2 mutant flies develop normally and are fertile, suggesting that these defects are mild and can be compensated by other mechanisms. In plants and other metazoans, it is similarly unclear whether RNA silencing can establish heterochromatin directly. In Arabidopsis thaliana, RNA-directed RNA polymerase, RDR2, a dicer protein, DCL3 and AGO4 mediate RNA-directed DNA methylation (RdDM), which has been implicated in centromeric repeat and TE silencing. However, H3K9me2 present in these regions is not lost in dcl3 and rdr2 mutants (Qi et al. 2006), a situation reminiscent of S. pombe siRNA-dependent heterochromatin assembly at the mating-type locus where a redundant pathway maintains heterochromatin formation in a manner independent of RNAi. A study in chicken demonstrated that Ago2 associate at very low levels with a constitutive heterochromatic domain that separates the 12 folate receptor gene and the ?-globin locus (Giles et al. 2010). The authors proposed that this heterochromatic region is maintained by a Dicer and Argonaute 2 dependent mechanism, however, it is not known whether this represents a general mechanism to maintain heterochromatin. Furthermore, a recent study in mouse cells proposed a model for a de novo HP1 targeting to pericentric heterochromatin by long non-coding RNAs corresponding to major satellites (Maison et al. 2011). Importantly, the authors did not detect any small dsRNA corresponding to these heterochromatic regions. POLYCOMB-MEDIATED GENE REPRESSION AND RNA SILENCING The highly conserved proteins of the two antagonizing complexes Polycomb group (PcG) and trithorax group (TrxG) maintain gene-expression profiles of crucial developmental regulators through either repression or activation, respectively. PcG/TrxG target genes possess cis-regulatory dual response elements termed Polycomb Response Elements (PREs), to which both PcG and TrxG proteins can be independently recruited by DNA-binding proteins (Simon and Kingston 2009). In Drosophila, in the silenced state, the DNA-binding protein Pleiohomeotic (PHO) (Klymenko et al. 2006) binds PREs and recruits Polycomb repressive complex 1 (PRC1) (Mohd-Sarip et al. 2005). PRC1- mediated ubiquitination in concert with PRC2-directed methylation of H3 at lysine 27(Wang et al. 2004) lead to Polycomb-i nduced gene silencing. Other DNA-binding proteins such as GAGA factor (GAF) have been suggested to function at PREs (Muller and Kassis 2006). During late embryogenesis, PcG and Tr xG proteins maintain the 13 repressed or activated state of the genes in homeotic clusters respectively by binding to PREs and modifying chromatin structure. RNA silencing has been suggested to affect the PcG response in a regulatory manner by influencing nuclear organization. A phenomenon, known as cosuppression, where multiple transgenic copies silence an endogenous gene, is sensitive to both PcG and RNA silencing components (Pal-Bhadra et al. 2002). Additionally, a transgenic reporter line that contains the Drosophila Fab-7 region, a PRE-containing boundary element that controls Abd-B expression, can induce PcG-dependent silencing of a reporter gene and of the endogenous scalloped (sd) gene (Bantignies et al. 2003). Argonaute mutants, ago1, piwi and aub were shown to disrupt Fab-7 PRE pairing-dependent silencing (Grimaud et al. 2006). Furthermore, RNA silencing has been suggested to play a role in PcG-dependent long-distance interaction between PRE-containing loci. Termed PcG bodies, these PRE-containing loci form approximately 50 nuclear foci that have been proposed to be sites of transcriptional repression that contain multiple PcG complexes bound to different PREs. Two remote PRE-containing loci have been shown to colocalize to the same PcG body and these long-range interactions were decreased in RNA silencing mutants. CHROMATIN INSULATORS, NUCLEAR ORGANIZATION AND RNA SILENCING It has become increasingly apparent that DNA topology is a critical determinant of gene regulation. While enhancers activate their target promoters over long distances, 14 insulators act to restrict these communications (reviewed in Wallace and Felsenfeld 2007). Chromatin insulators are defined as DNA-protein complexes that are characterized by two functional properties, enhancer blocking and barrier activity (Figure 1-3). In the former case, a chromatin insulator, positioned between an enhancer and a promoter, can interfere with enhancer-promoter interactions in a directional manner. In the latter case, an insulator can buffer transgenes against the spread of silent chromatin. Figure 1-3. Functional properties of chromatin insulators. A) Enhancer blocking activity of chromatin insulators restricts enhancer-promoter communications while (B) barrier function demarcates silent and active chromatin domains. Unlike vertebrates, which possess only one known insulator protein, the CCCTC- binding factor (CTCF) (Phillips and Corces 2009), Drosophila employs at least five known insulators defined by their DNA binding proteins. Two well-characterized insulators are the gypsy insulator and Frontabdominal-8 (Fab-8) (Moon et al. 2005; Gerasimova et al. 2007; Mohan et al. 2007) each containing binding sites for the zinc- finger DNA binding proteins Suppressor of Hairy wing (Su(Hw)) and CTCF, respectively A. Heterochromatin Insulator Enhancer Insulator Promoter B. 15 (Figure 1-4). The gypsy insulator contains an additional component, Mod(mdg4)2.2, a BTB domain protein that directly interacts with Su(Hw) but not DNA. The two insulators share a common component, Centrosomal protein 190 (CP190) (Pai et al. 2004; Gerasimova et al. 2007), which inter acts with Su(Hw), Mod(mdg4)2.2 and CTCF. Despite thousands of insulator binding sites throughout the genome, insulator complexes localize to a few large nuclear foci termed insulator bodies. These bodies are proposed to be insulator sites clustered together to organize chromatin into distinct transcriptional domains, and the integrity of insulator bodies is highly correlated with gypsy insulator function (Figure 1-4; reviewed in Bushey et al. 2008). It has been proposed that chromatin insulators interact with each other or with other cis-regulatory elements to form chromatin loops. Chromosome conformation capture (3C) studies have demonstrated an interaction between two insulator sites (Blanton et al. 2003). Moreover, gypsy insulator loops have been visualized in salt-extracted nuclei by fluorescence in situ hybridization (Byrd and Corces 2003). 16 A. C. Insulator body gypsy Mod(mdg4)2.2 Su(Hw) CP190 B. Fab-8 CTCF CP190 g ypsy insulator complex CTCF/CP190 insulator complex Figure 1-4. Nuclear organization of a chromatin insulator. A. The gypsy insulator is defined by its binding protein Su(Hw), which also interacts with Mod(mdg4)2.2 and CP190. B. The Fab-8 insulator, located in the Abd-B locus of the BX-C complex, is bound by CTCF and CP190. C. Insulator proteins colocalize in large nuclear structures termed insulator bodies. It has been proposed that insulator complexes come together to establish transcriptional domains by looping of the DNA. The Fab-8 insulator is a part of a large homeotic gene ( Hox ) cluster, known as the bithorax complex (BX-C), that controls the identity of nine parasegments (PS5-14) in the posterior two-thirds of the fly by regulating the expression of three homeotic genes, Ultrabithorax ( Ubx ), abdominal-A (abd-A) and Abdominal-B (Abd-B) (Figure 1-5). Each parasegment-specific infraabdominal (iab) domain contains an enhancer that is kept 17 autonomous by boundary elements, Mcp , Fab-6, Fab-7 and Fab-8 (reviewed in Maeda and Karch 2009). For example, in early embryogenesis, Abd-B expression is controlled by enhancers in iab-5-8 cis-regulatory domains in PS10-13. CTCF is present at Mcp , Fab-6, and Fab-8 boundary elements and has been shown to be required for Fab-8 insulator function (Moon et al. 2005 ; Holohan et al. 2007). Moreover, Fab-8 interacts with a region bound by CTCF near the Abd-B promoter (Kyrchanova et al. 2008), suggesting that boundary elements regulate proper communication between enhancers and the Abd-B promoter via CTCF organizing chromatin domains at BX-C. Insulators and other cis-regulatory regions in the Abd-B locus engage in numerous long-range interactions, and the precise topology of the locus has been postulated to be a central mechanism of tissue-specific Abd-B regulation (Cleard et al. 2006; Lanzuolo et al. 2007; Kyrchanova et al. 2008; Bantignies et al. 2011). However, the mechanism by which chromosome looping is achieved at this locus has not been elucidated. Vertebrate CTCF has been demonstrated to mediate chromosomal looping at several developmentally regulated loci in concert with cohesin (reviewed in Merkenschlager 2010), but it is not known whether Drosophila CTCF, which only shares homology in the zinc-finger DNA binding domain, retains the capacity to promote looping. Lastly, a recent genome-wide study in mammalian cells revealed that CTCF mediates numerous promoter-enhancer communications suggesting a role for CTCF that is diverse from its enhancer-blocking function (Handoko et al. 2011). 18 Figure 1-5. Abd-B cis -regulatory region of the bithorax complex (BX-C). A schematic representation of iab-2 through iab-8 cis-regulatory domains that encompass two transcription units: abd-A and Abd-B, arrows indicate the direction of transcription. Boundary elements, Mcp , Fab-6, Fab-7 , and Fab-8 and PREs are also indicated. The corresponding abdominal segments specified by the iab domains are shown in the Drosophila embryo. Modified from Akbari et al., Dev. Biol., 2006. Several observations implicate RNA silencing in insulator function. First, Rm62, a DEAD-box putative RNA helicase required for dsRNA-mediated silencing copurifes with CP190 insulator complexes from Drosophila nuclear embryonic extract in an RNase A-sensitive manner (Lei and Corces 2006). Second, genetic analysis of Rm62 mutants revealed improved activity of the gypsy insulator while mutations in Argonaute genes, piwi and aub, cause decreased gypsy insulator function. Third, microscopic examination of insulator bodies in larval imaginal discs of Rm62, piwi and aub mutants revealed 19 disruption of insulator body organization. Insulator body phenotypes correlated with gypsy insulator activity, suggesting that RNA silencing plays a role in nuclear organization of gypsy insulator complexes. Whether RNA silencing can affect chromatin insulator activity of an insulator other than gypsy has not been determined. CONCLUSION It has become increasingly apparent that long-range chromosomal interactions driven by cis-regulatory elements are critical for proper control of gene expression. Chromatin insulators disrupt enhancer-promoter interactions and can protect transgenes from the effects of silent chromatin exerted by heterochromatin or PcG-induced repression. In Drosophila, both the gypsy chromatin insulator and PREs have been postulated to mediate long-range interactions to promote higher order chromatin organization. These long-range interactions are, however, perturbed in RNA silencing mutants. Furthermore, a functional link has been reported between the gypsy insulator, acting to restrict PRE-mediated chromatin looping, and PcG-induced silencing (Comet et al. 2011). The plethora of evidence suggests that RNA silencing has a functional role in higher order chromatin organization. The mechanistic details regarding the interaction between RNA silencing components and insulator proteins along with PREs are, however, missing. Here, I investigate whether RNA silencing affects gene expression at the level of higher order chromatin organization. For my studies I utilize Drosophila melanogaster, an outstanding model organism that has a short life cycle, a large array of available 20 genetic tools, a sizable pool of characterized mutants and cell lines. Specifically, I investigate two aspects of RNA involvement in chromatin function; (1) whether a role for RNA silencing in heterochromatin nucleation and maintenance is conserved in Drosophila, and (2) elucidate how RNA silencing affects the function of the CTCF class of chromatin insulators. First, I examine the effects of piRNA and endo-siRNA silencing mutants on heterochromatin recruitment to the sites of piRNA and endo-siRNA production utilizing genetic and biochemical approaches. Second, I investigate the role of AGO2 on CTCF/CP190-dependent Fab-8 insulator function, PREs and active promoters, and its involvement in nuclear organization. Overall, these studies will shed more light on mechanistic details of RNA silencing function in higher order chromatin organization. 21 CHAPTER 2 HP1 RECRUITMENT IN THE ABSENCE OF ARGONAUTE PROTEINS IN DROSOPHILA ABSTRACT Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo- siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Contrary to expectation, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site suggesting indirect effects on HP1 recruitment. Taken 22 together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways. 23 INTRODUCTION Heterochromatin, characterized by scarcity of genes, low levels of transcription, late replication, and low recombination rates, maintains genomic stability by hindering propagation of potentially deleterious transposable elements (TEs). One model for heterochromatin assembly comes from studies in fission yeast where a role for RNA interference (RNAi), a process by which dsRNAs silence the expression of target genes, was demonstrated (reviewed in (Grewal and Elgin 2007)). Based on the model in S. pombe, where transcription from pericentromeric repeat-rich regions initiates heterochromatin formation via interaction with the RNA silencing machinery, it has been suggested that the mechanism may be evolutionarily conserved between unicellular eukaryotes and metazoans. In the Drosophila genome, there is an extensive overlap between TE-rich regions and pericentromeric and telomeric heterochromatin marked by non-histone Heterochromatin Protein 1 (HP1) and histone 3 dimethylated at lysine 9 (H3K9me2). The phenomenon of position-effect variegation (PEV) provided the first glimpse into the role of heterochromatin in gene silencing in Drosophila. When a normally euchromatic gene is relocated near heterochromatin, variegated expression results from variable levels of heterochromatin spreading over the gene in each cell. Screens for dominant mutations that either suppress or enhance { Enhancer of variegation [ E(var) ] } PEV were performed to identify key components of heterochromatin. For example, mutation of Su(var)3-9 , which encodes an H3K9 methyltransferase, was identified in a large screen for modifiers of PEV (Tschiersch et al. 1994). A ccordingly, loss of HP1, encoded by Su(var)2-5, 24 causes increased expression of a gene subject to PEV, while an extra copy has the reverse effect (Eissenberg et al. 1990). Sequencing of the small RNA population associated with the Piwi claude of AGO proteins, Piwi, Aub and AGO3 from Drosophila ovaries identified a subclass of ~24-30 nt long RNAs, termed Piwi-interacting RNAs (piRNAs) (Brennecke et al. 2007; Yin and Lin 2007). Interestingly, piRNAs originate from discrete loci, the majority of which are located at pericentromeric and telomeric regions containing TEs and other repetitive elements. Termed piRNA clusters, these loci range in length between a few of to hundreds Kb. Two prominent clusters include the 42AB piRNA locus on chromosome 2R and flamenco (flam) piRNA producing locus on the X chromosome. The flam locus was originally discovered as a region involved in the control of retrotransposons: gypsy, Idefix , and ZAM (Prud'homme et al. 1995; Desset et al. 2003). Examination of piRNAs associated with Piwi, Aub and AGO3 revealed nucleotide signatures indicative of the amplification ?ping-pong? cy cle (Brennecke et al. 2007; Gunawardane et al. 2007). Aub and AGO3, which both possess the Slicer endonuclease activity, are cytoplasmic proteins that most likely silence TEs post-transcriptionally in the germ cells. Interestingly, in the ovarian somatic cell (OSC) line, where Piwi is highly expressed but not Aub or AGO3, Piwi-associated piRNAs that are mostly derived from flam locus are derived by the primary processing pathway that does not involve the amplification step (Saito et al. 2009). Piwi, a nuclear protein that is present in both somatic and germ cells of the ovary, is required for the TE silencing in gonadal somatic cells. Thus, it is possible that Piwi can recruit heterochromatin machinery to silence the repetitive elements in the nucleus. 25 The role of Piwi and the somatic primary piRNAs as epigenetic regulators remains controversial since Piwi seems to exert the opposite effects at different genomic sites. On one hand, piwi mutants exhibit defects in heterochromatic silencing and heterochromatin localization, and Piwi and HP1 interact physically and associate with heterochromatic 1360 and F element transposable elements, suggesting that Piwi and the somatic primary piRNAs may silence TEs by recruiting heterochromatin (Pal-Bhadra et al. 2004; Vagin et al. 2006; Brow er-Toland et al. 2007; Malone et al. 2009). On the other hand, piwi mutants show both increased transcriptional silencing and HP1 association with the 3R-TAS subtelomeric region, indicating an epigenetic activation role. Therefore, it remains an open question whether the piRNA pathway can recruit heterochromatin to its target sites. Sequencing of small RNAs associated with AGO2 revealed a population of ~21- 22 nt long species in Drosophila embryos, ovaries and S2 embryonic cells termed endogenous siRNAs (endo-siRNAs) (Chung et al. 2008; Czech et al. 2008; Ghildiyal et al. 2008; Kawamura et al. 2008). The majority of endo-siRNAs exhibit homology to TEs and other repetitive sequences and overlap considerably with piRNA clusters. Studies have reported HP1 mislocalization defects in early embryos and on larval polytene chromosomes in ago2 mutants suggesting endo-siRNA pathway involvement in heterochromatin formation (Deshpande et al. 2005; Fagegaltier et al. 2009). Whether the endo-siRNA pathway can directly recruit HP1 to heterochromatin remains unclear. In this study, I investigated whether HP1 association with heterochromatin in Drosophila is mediated by either the piwi dependent piRNA pathways or by the AGO2 dependent endo-siRNA pathway. I hypothesized that similarly to the RNAi-dependent 26 heterochromatin recruitment in fission yeast, heterochromatin assembly depends on piRNA and endo-siRNA pathways that silence TEs in the germline and soma respectively. In order to address this hypothesis, I examined heterochromatin localization as marked by HP1 to piRNA producing loci in piRNA and endo-siRNA pathway mutants genetically, utilizing transcriptional reporters, and biochemically by Chromatin Immunoprecipitation (ChIP). Transcriptional reporters bearing mini-white transgene and positioned inside or in close proximity to top piRNA producing loci, flam, 42AB , and 80EF on chromosome 3L were used to measure mini-white expression in the adult eye. ChIP, an immunoprecipitation technique used to determine whether specific proteins are associated with specific genomic regions in vivo, was utilized to assay HP1 localization to piRNA clusters. Specifically, I hypothesized that if heterochromatin recruitment depends on piRNA and/or endo-siRNA silencing pathways then a disruption in Piwi and AGO2 proteins would result in (a) decreased silencing as measured by transcriptional reporters at the piRNA producing loci and (b) a decrease of HP1 association with piRNA clusters. Here, I show that piRNA clusters are subject to heterochromatic silencing and bound by HP1. Contrary to expectation, mutation of AGO2, piwi or aub results in increased silencing at piRNA clusters and an increase in HP1 association with these loci. Furthermore, loss of piRNA production at a single piRNA locus results in global redistribution of HP1 and a reduction of silencing at a distant heterochromatic site. Therefore, our results indicate that HP1 can associate with chromatin independently of both endo-siRNA and piRNA silencing pathways in the soma. 27 RESULTS Heterochromatin dependent transcriptional silencing at piRNA clusters We sought to determine if HP1 is recruited to heterochromatin by AGO2 or Piwi. The majority of genomic regions that produce the bulk of piRNA, termed piRNA clusters, are pericentromeric and rich in transposable elements (Brennecke et al. 2007; Yin and Lin 2007). These re gions also produce endo-siRNA (Chung et al. 2008; Czech et al. 2008; Ghildiyal et al. 2008; Kawamura et al. 2008), and due to their proximity to the centromere, may be heterochromatic and serve as platforms for Argonaute mediated HP1 recruitment. In order to test genetically whether pericentromeric piRNA clusters are heterochromatic, we examined a collection of fly lines bearing P element transgene insertions inside or in close proximity to four piRNA producing loci, flam, 80EF, 42AB , and 38C . The P elements contain a mini-white transcriptional reporter that was assayed for expression in the adult eye. Genomic locations of these transgene insertions are indicated in relation to previously identified small RNAs immunoprecipitated with Piwi, Aub/AGO3, and AGO2 respectively from various cell types (Figure 2-1) (Brennecke et al. 2007; Yin and Lin 2007; Czech et al. 2008; Kawamura et al. 2008). Lines harboring P elements inside or in the vicinity of a piRNA cluster exhibit variegating coloration of distinct eye facets similar to PEV, sugges ting the presence of variably spreading heterochromatin at their sites of insertion (Figure 2-2, Table 2-1). Interestingly, insertions within a piRNA cluster that display high mini-white expression without 28 variegation harbor SUPor-P constructs, which contain Suppressor of Hairy wing [Su(Hw)] insulator sequences that flank and likely protect the mini-white reporter from the effects of surrounding heterochromatin (Roseman et al. 1993). 29 Figure 2-1. Schematic representation of four top piRNA clusters. Genomic locations of small RNAs, primer sets used for ChIP, and P element insertions at (A) flam piRNA cluster on chromosome X, (B) 80EF piRNA cluster on chromosome 3L, (C) 42AB piRNA cluster on chromosome 2R and (D) 38C piRNA cluster on chromosome 2L. Sequence datasets derived from previous studies were mapped to the genome using Bowtie software allowing two or zero mismatches (Langmead et al. 2009). Piwi- immunoprecipitated (Brennecke et al. 2007; Yin and Lin 2007), Aub or AGO3- immunoprecipitated (Brennecke et al. 2007) and AGO2-immunopreci pitated (Czech et al. 2008; Kawamura et al. 2008) reads mapping to multiple locations in the genome are indicated in red (with two mismatches allowed) and pink (with zero mismatches allowed) while uniquely mapping reads are in dark blue (with two mismatches allowed) and light blue (with zero mismatches allowed). Primer sets used for ChIP analysis are indicated by yellow arrowheads. Strongly variegating (dark green triangle), weakly variegating (light green triangle), and non-variegating P elements with high expression levels (white triangle) are indicated. (A) P{EPgy2}DIP1 EY02625 , (B) PBac(PB) c06482 , and (C) P{EPgy2}EY08366 P element insertions are marked by an asterisk. SUPor-P P elements containing insulator sequences are marked by an ?I?. Centromere proximal end is marked by a hollow C. RepeatMasker detected sequences are represented in black. 30 31 Table 2-1. Expression of mini-white in fly lines harboring P element insertions in four top piRNA clusters. Insertion piRNA cluster Genomic coordinates of insertion Variegation Inside piRNA cluster P{EPgy2}DIP1 EY02625 flam X:21,501,171 [-] Yes No P{SUPor-P}flam KG00476 flam X:21,505,285 [-] No No P{GT1}flam BG02658 flam X:21,502,538 [-] Yes No PBac(WH)CG32230 f00651 80EF 3L:23,237,018 [+] Weak No PBac(PB) c06482 80EF 3L:23,286,922 [-] Yes Yes PBac(PB)CG40470 c06318 80EF 3L:23,849,420 [+] No No P{GT1}BG01672 BG01672 42AB 2R:2,370,529 [-] No Yes P{EPgy2}EY08366 42AB 2R:2,129,510 [+] Yes No P{XP}d02126 42AB 2R:2,129,452 [+] Weak No P{SUPor-P}Pld KG02714 42AB 2R:2,133,438 [+] No No P{SUPor-P}KG09351 42AB 2R:2,160,357 [-] No Yes PBacf(WH)04291 42AB 2R:2,228,280 [-] Weak Yes P{EPgy2}EY01034 38C 2L:20,205,306 Yes Yes P{XP}d02757 38C 2L:20,174,988 [+] Weak Yes PBac(WH)f03348 38C 2L:20,165,746 Weak Yes P{SUPor-P}KG05288 38C 2L:20,166,034 [+] No Yes PBac{RB}e03575 38C 2L:20,121,359 [+] Weak No P{SUPor-P}KG02342 38C 2L:20,120,504 [-] No No The genomic coordinates for four top piRNA clusters were defined as previously determined by Brennecke et al., 2007. The genomic coordinates of the P-element insertions were confirmed by PCR with primers specific to the P-elements and flanking genomic sequences. 32 Expression analysis of these transcriptional reporter insertions indicates that piRNA clusters and their immediate vicinity are subject to HP1 dependent silencing. Reporter expression levels of three lines harboring an insertion at flam, 80EF, or 42AB with the most apparent variegation were tested for dependence on heterochromatin. P{EPgy2}DIP1 EY02625 is inserted in a gene located on the centromere distal side of the flam piRNA producing locus on the X chromosome (Figure 2-1A), PBac(PB)c06482 resides within the 80EF cluster on chromosome 3L (Figure 2-1B), and P{EPgy2}EY08366 borders the centromere proximal edge of the 42AB piRNA locus on chromosome 2R (Figure 2-1C). In order to test whether these reporters are sensitive to perturbation of heterochromatin, the expression of mini-white was examined in Su(var)2- 505/+ and Su(var)3-9 1/ + dominant loss-of-function mutants, which are compromised for HP1 and H3K9 methyltransferase activity respectively. As expected, decreased silencing of mini-white expression resulting in increased pigmentation was observed for all three insertions in the heterochromatin mutants compared to wild type (Figure 2-2), suggesting that the vicinity of P element insertion are indeed heterochromatic. 33 Figure 2-2. piRNA and endo-siRNA pathway mutants display increased silencing of transcriptional reporters at or near piRNA clusters. Adult eyes of wild type, Su(var)2-5 05/+ , Su(var)3-9 1/+ , piwi1/piwi2, aubQC42 /aub?P-3a , and/or AGO2 - /AGO2 - mutants carrying a mini-white transgene inserted inside or in close proximity to the (A) flam, (B) 80EF, and (C) 42AB piRNA clusters. AGO251B/AGO2414 mutants are examined in (A) while AGO251B mutants are examined in (C). (D) Levels of eye pigment measured at 480 nm extracted from male heads of the indicated genotypes (A-C). 34 Su(var)2-505/+ Su(var)2-505/+ Su(var)2-505/+ Su(var)3-91/+ Su(var)3-91/+ Su(var)3-91/+ aubQC42/aub?P-3a aubQC42/aub?P-3a aubQC42/aub?P-3a piwi1/piwi2 piwi1/piwi2 AGO251B WT WT WT fla m 80E F 42AB AGO251B/AGO2414 piwi1/piwi2 0.2 0.4 0.6 0.1 0.2 0.3 0.4 0.2 0.4 0.6 0.8 Su(var)2- 5 05 /+ Su(var)3- 9 1 /+ au b QC4 2 /au b ?P-3a AGO2 - /AGO2 - WT piw i1 /piw i2 flam 80EF 42AB OD 480 A B C D 35 piRNA and endo-siRNA pathway mutants decrease transcription at piRNA clusters We next tested whether the transcriptional reporters at piRNA clusters are sensitive to perturbations in the piRNA and endo-siRNA silencing pathways. If Piwi were responsible for direct recruitment of HP1 to piRNA clusters, mutation of piwi should increase mini-white expression similarly to disruption of heterochromatin. Surprisingly, piwi1/piwi2 loss-of-function mutants exhibit a substantial loss of reporter expression indicating increased silencing when compared to wild type (Figure 2-2). Furthermore, aubQC42 /aub?P-3a loss-of-function piRNA pathway mutants result in a similar reduction of mini-white expression. Strikingly, the flam transcriptional reporter expression level was decreased dramatically in the transheterozygous endo-siRNA pathway mutant, AGO251B/AGO2414 compared to wild type (Figure 2-2A). Similarly, in the AGO251B null mutant, the 42AB transcriptional reporter displays almost complete silencing (Figure 2-2C). Spectroscopic analysis of extracted eye pigment verifies the overall changes in mini-white expression levels for each genotype compared to wild type (Figure 2-2D). Additionally, examination of Dcr-2L811fsX mutants shows a similar mild increase in silencing for the transcriptional reporter inserted near flam (Figure 2-3A). The opposite effects of piRNA and endo-siRNA pathway mutations compared to heterochromatin mutations suggest that these RNA silencing pathways may actually oppose heterochromatin formation at piRNA clusters. 36 Figure 2-3. Dcr-2 mutants display increased HP1 chromatin association and increased silencing at piRNA clusters. ChIP at (A) flam and (B) 80EF piRNA clusters in wild type (blue) and Dcr-2L811fsX /+ (orange) from adult heads with antibodies specific to HP1. Values shown are percent input immunoprecipitated for each primer set normalized to hsp26. Error bars indicate standard deviation of quadruplicate PCR measurements. (C) Adult eyes of wild type and Dcr-2L811fsX mutants carrying a mini- white transgene inserted in close proximity to the flam piRNA cluster. 37 0 1 2 3 4 5 6 7 hs p2 6 ye llow TART 13 6 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Enrichment relative to hsp2 6 0 1 2 3 4 5 6 7 h s p2 6 y ell o w T A RT 13 6 0 A B C D E F G H I J K L M N O P Primer sets WT Dcr-2 L811fsX Primer sets ?-HP1 ?-HP1 A B WT Dcr-2 L811fsX WT fla m Dcr-2 L811fsX C flamenco 80EF Enrichment relative to hsp2 6 38 HP1 chromatin association is increased at piRNA clusters in somatic tissues of RNA silencing mutants In order to further examine the heterochromatic nature of piRNA clusters at higher resolution, ChIP assays were performed in adult heads to assess HP1 association with two piRNA clusters, flam and 80EF, in the soma. Genomic locations of primer sets that uniquely amplify regions spanning these piRNA clusters are indicated in Figure 2- 1A-B. As positive controls, primers for two transposable elements known to recruit HP1, TART, a telomere-specific non-LTR retrotransposon, and 1360, a DNA transposon, were also tested (Fanti et al. 1998; S un et al. 2004). Euchromatic genes hsp26 and yellow were also included in the analysis as negative controls for HP1 association. In wildtype fly heads, HP1 is observed at or near locations that give rise to piRNAs and endo-siRNAs at both flam and 80EF loci. ChIP was performed using ?- HP1 antibodies in chromatin prepared from wildtype heads, and the amount of DNA associated was determined by quantitative PCR using specific primer sets. As expected, low levels of hsp26 and yellow are immunoprecipitated with HP1, while TART and 1360 levels are enriched above the euchromatic genes by over six-fold (Figure 2-4). At flam, HP1 associates with the majority of regions that produce high levels of piRNAs or endo- siRNAs approximately two to three-fold over the euchromatic sites (Figure 2-4A, primer sets 1-15). Similarly, at 80EF, HP1 immunoprecipitates piRNA and endo-siRNA producing regions two to three-fold higher than the negative controls indicating the presence of heterochromatic marks at these loci (Figure 2-4B, primer sets G-M). Regions flanking these areas display approximately one to two-fold enrichment over euchromatic 39 sites, which may be due to tapering of HP1 spreading (Figure 2-4B, primer sets A-F and N-P). ChIP using antibodies directed against the chromatin insulator protein Su(Hw) verified its presence at known insulator sequences gypsy and 1A-2 (Parnell et al. 2003) but only background levels at TART, 1360, and piRNA clusters, i ndicating the specificity of HP1 association at these sites (Figure 2-5). Rabbit IgG negative control immunoprecipitations yielded negligible amounts of DNA for all sites tested (<0.3% input). Consistent with the transcriptional reporter assay, RNA silencing mutants display elevated levels of HP1 at piRNA clusters. ChIP of HP1 was performed in piwi1/piwi2 mutant heads, and similar levels at positive and negative controls were obtained compared to wild type (Figure 2-4). In contrast, at the flam locus, a two to five-fold increase in HP1 levels is observed at the centromere proximal side of the locus compared to wild type (Figure 2-4A, primer sets 6- 15). Little change in HP1 recruitment is observed at the centromere distal end of flam in piwi1/piwi2 mutants (Figure 2-4A, primer sets 1-5). At 80EF, HP1 levels increase two to three-fold in piwi1/piwi2 mutants compared to wild type across all primer sets examined (Figure 2-4B, primer sets A-P). In order to address differences in strain background and potential accumulation of TEs in piwi mutant strains, we performed ChIP assays comparing piwi1/piwi2 mutants to a piwi1/ + heterozygous strain and obtaine d similar results (Figure 2-6). ChIP experiments performed in AGO251B mutant heads show a similar overall increase of HP1 at piRNA clusters compared to piwi1/piwi2 mutants. Levels of HP1 at hsp26, yellow, TART, and 1360 are similar in AGO251B mutants and wild type while differences are apparent at piRNA clusters (Figure 2-4). At flam, AGO251B mutants 40 display a two to seven-fold increase of HP1 association with the centromere proximal side compared to wild type (Figure 2-4A, primer sets 6-15). At th e centromere distal end, no significant changes in HP1 levels are detected (Figure 2-4A, primer sets 1-5). For 80EF, AGO251B mutants show similar levels of HP1 to wild type at the centromere distal end (Figure 2-4B, primer sets A-D) while an approximately two to five-fold increase of HP1 is detected in the remainder of the regions tested (Figure 2-4B, primer sets E-P). Moreover, ChIP assays in AGO251B homozygous mutants compared to an AGO251B/+ heterozygous strain produced similar results (Figure 2-7). Similar to AGO251B mutants, Dcr-2L811fsX mutants show an increase of HP1 at regions that produce small RNAs compared to wild type (Figure 2-3 (B-C)). HP1 protein levels in wildtype, piwi1/piwi2, and AGO251B fly heads are similar indicating that the increased chromatin association observed is not due to an increased amount of HP1 (Figure 2-8). The increased HP1 chromatin association with piRNA clusters in RNA silencing mutants compared to wild type is consistent with increased silencing of P element insertions, and these results suggest that at least some of the observed effects on reporter gene expression in RNA silencing mutants are due to chromatin related events. Taken together, these data suggest an antagonistic effect of Piwi, Aub, and AGO2 on HP1 recruitment to chromatin in somatic tissue. 41 0 2 4 6 8 10 12 14 hs p2 6 ye llow TART 13 6 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 % Input 0 2 4 6 8 10 12 14 16 18 hs p2 6 ye llow TART 13 6 0 A B C D E F G H I J K L M N O P % Input A B Primer sets Primer sets ?-HP1 ?-HP1WT piwi1/piwi2 AGO251B WT piwi1/piwi2 AGO251B flamenco 80EF Figure 2-4. HP1 associates with chromatin at piRNA clusters, and its levels increase in RNA silencing mutants. ChIP at (A) flam and (B) 80EF piRNA clusters in wild type (blue), piwi1/piwi2 (yellow), and AGO251B (red) mutants from adult heads with antibodies specific to HP1. Percent input immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. 42 0 0.2 0.4 0.6 0.8 1 1.2 1.4 hs p 26 ye l l ow TART 13 6 0 gy p sy 1A - 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 % Input 0 0.2 0.4 0.6 0.8 1 1.2 1.4 hs p 26 ye l l ow TART 13 6 0 gy p sy 1A - 2 A B C D F G H I J K L M N O P % Input A B Primer sets Primer sets flamenco 80EF Figure 2-5. Su(Hw) does not associate with chromatin at piRNA clusters in heads. ChIP at (A) flam and (B) 80EF piRNA clusters in wild type with antibodies specific to Su(Hw) (blue) and rabbit normal serum (yellow). Percent input immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. ?-Su(Hw) IgG ?-Su(Hw) IgG 43 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 piwi1/+ piwi1/piwi2 Enrichment normalized to hsp2 6 Enrichment normalized to hsp2 6 A B ?-HP1 ?-HP1 flamenco 80EF hs p2 6 ye llow TART 13 6 0 gy psy m dg1 A B C D E F G H I J K L M N O P Primer sets h sp 26 yello w T A RT 136 0 g yp sy m d g1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Primer sets piwi1/+ piwi1/piwi2 Figure 2-6. HP1 chromatin association levels are increased in piwi mutants at piRNA clusters. ChIP at (A) flam and (B) 80EF piRNA clusters in piwi1/+ (light grey) and piwi1/piwi2 (dark grey) from adult heads with antibodies specific to HP1. Values shown are percent input immunoprecipitated for each primer set normalized to hsp26. Error bars indicate standard deviation of quadruplicate PCR measurements. 44 0 0.5 1 1.5 2 2.5 3 3.5 4 h s p2 6 y e llo w T A R T 13 6 0 g y ps y m d g 1 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 Enrichment normalized to hsp2 6 0 0.5 1 1.5 2 2.5 3 3.5 4 h s p2 6 y e llo w T A R T 13 6 0 g y ps y m d g 1 A B C D E F G H I J K L M N O P A B Primer sets Primer sets ?-HP1 ?-HP1 flamenco 80EF AGO251B /+ AGO251B AGO251B /+ AGO251B Enrichment normalized to hsp2 6 Figure 2-7. HP1 chromatin association levels are increased in AGO2 mutants at piRNA clusters. ChIP at (A) flam and (B) 80EF piRNA clusters in AGO251B/+ (light blue) and AGO251B (dark blue) from adult heads with antibodies specific to HP1. Values shown are percent input immunoprecipitated for each primer set normalized to hsp26. Error bars indicate standard deviation of quadruplicate PCR measurements. 45 fla m 1 WT AGO 2 51 B piw i1 /piw i2 Pep HP1 Figure 2-8. HP1 protein levels in wildtype, flam1 , AGO2 51B and piwi 1 /piwi 2 fly heads. Total protein was extracted from twenty adult heads by homogenization in RIPA buffer and separated by SDS-PAGE. Immunoblotting of HP1 and Protein on Ecdysone Puffs (Pep), a nuclear protein serving as a loading control, is shown. 46 HP1 also associates with piRNA clusters in ovaries Given the evidence that transposable elements are mainly silenced in the gonad via piRNA pathways and in the soma via the endo-siRNA pathway, we wanted to determine whether HP1 also associates with piRNA clusters in gonadal tissues. Therefore, we investigated HP1 recruitment to piRNA clusters in wildtype ovaries by ChIP. As in heads, low levels of hsp26 and yellow are immunoprecipitated with HP1, whereas TART and 1360 levels are enriched above th e euchromatic genes by over ten- fold (Figure 2-9). At the flam locus, a four to fifteen-fold increase over the euchromatic sites in HP1 levels is observed at most sites at the centromere proximal side of the locus (Figure 2-9A, primer sets 4-15). Similarly, at 80EF, HP1 immunoprecipitates small RNA producing regions two to twenty-fold higher than euchromatic sites indicating the presence of heterochromatic marks at these loci (Figure 2-9B, primer sets A-P). Rabbit IgG negative control immunoprecipitations yielded negligible amounts of DNA for all sites tested. We were unable to immunoprecipitate DNA at levels above background from either heads or whole ovaries using multiple antibodies to Piwi, Aub, AGO3, and AGO2 that have been used in previous studies for immunoprecipitation or immunofluorescence (data not shown; Miyoshi et al. 2005; Saito et al. 2006; Brower- Toland et al. 2007; Guna wardane et al. 2007). 47 A B ?-HP1 0 5 10 15 20 25 30 % Input HP1 IgG 0 5 10 15 20 25 30 35 % Input HP1 IgG ?-HP1 hs p2 6 ye llow TART 13 6 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Primer sets hs p2 6 ye llow TART A B C D F G H I J K L M N O P Primer sets flamenco 80EF 13 6 0 Figure 2-9. HP1 associates with chromatin at piRNA clusters in ovaries. ChIP at (A) flam and (B) 80EF piRNA clusters in wildtype ovaries with antibodies specific to HP1 (blue) and normal rabbit IgG (red). 48 HP1 chromatin association is not affected greatly by depletion of Piwi in somatic ovarian follicle cells We wished to address whether HP1 association with piRNA clusters is dependent on Piwi in the gonad, which express high levels of both proteins. Due to a complete loss of germ cells and the severe underdevelopment of ovary tissue in piwi mutants, it was not possible to obtain enough mutant material to perform ChIP. Therefore, we examined the recruitment of HP1 to chromatin in an ovarian somatic follicle cell line (OSC) that expresses Piwi but not Aub or AGO3 and produces only primary piRNAs, a large proportion of which derive from the flam locus (Saito et al. 2009). The majority of Piwi was depleted from OSC cells by siRNA-mediated knockdown, and depletion of Piwi does not affect HP1 or Lamin protein levels compared to mock transfected cells (Figure 2-10A). Subsequently, we investigated HP1 recruitment to piRNA clusters by ChIP in OSC cells. In mock treated cells, low levels of hsp26 and yellow are immunoprecipitated with HP1, while TART and 1360 levels are enriched above the euchromatic genes by 1.5- to over two-fold (Figure 2-10(B-C)). Two additional TEs tested, gypsy and mdg1, are immunoprecipitated at similar levels to TART with HP1 (Figure 2-10(B-E)). At flam, HP1 associates with the piRNA cluster similar to TE levels (Figure 2-10(B-C)). Despite much lower piRNA production from the 80EF cluster in OSC compared to flam (Saito et al. 2009), HP1 associates with piRNA producing regions of 80EF at similar levels to flam and TEs (Figure 2-10C, primer sets A-P). Overall, the HP1 recruitment profile in OSC is similar to that of heads and whole ovaries albeit at lower relative levels. In Piwi 49 knockdown cells, no significant differences are seen for HP1 recruitment to all sites compared to mock treated cells except a two-fold decrease at the 1360 element. Rabbit IgG negative control immunoprecipitations yielded low amounts of DNA for all sites tested (<0.06% and <0.07% input for mock and Piwi knockdown cells, respectively). Importantly, Piwi association with chromatin is detectable in OSC cells, but its profile differs from that of HP1. In mock treated cells, antibodies directed against Piwi (Saito et al. 2006) immunoprecipita te euchromatic sites at levels similar to that of TEs (Figure 2-10(D-E)). Furthermore, the majority of regions producing piRNA at flam is also immunoprecipitated at comparable levels to both euchromatic sites and TEs (Figure 2-10D). Moreover, levels of Piwi association with 80EF is akin to that of flam, while several sites in both flam and 80EF clusters show particular enrichment of Piwi up to three-fold compared to the average association with other sites tested (Figure 2-10(D-E)). In Piwi knockdown cells, Piwi chromatin association drops two to five-fold, down to background levels at all sites except for some residual association with two sites in or near the flam locus. Mouse IgG negative control immunoprecipitations yielded low amounts of DNA in comparison to ?-Piwi immunoprecipitations in mock treated cells for all sites tested (<0.04% and <0.02% i nput for mock and Piwi knockdown cells, respectively). We conclude that in ovarian somatic follicle cells, reduction of the total pool of Piwi as well as the chromatin bound fraction does not affect HP1 association with piRNA clusters and has a minimal effect on HP1 association with TE chromatin association. 50 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 hsp2 6 yellow TA R T 136 0 gypsy mdg 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 % Input 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 hsp2 6 yellow TA R T 136 0 gypsy mdg 1 A B C D E F G H I J K L M N O P 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 ?-Piwi, Mock ?-Piwi, Piwi KD hsp2 6 yellow TA R T 136 0 gypsy mdg 1 A B C D E F G H I J K L M N O P A Mock Piwi KD Piwi Lamin HP1 B C D E Primer sets Primer setsPrimer sets flamenco 80EF 80EF % Input % Input % Input ?-HP1, Mock ?-HP1, Piwi KD 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Primer sets flamenco ?-Piwi, Mock ?-Piwi, Piwi KD hsp2 6 yellow TA R T 136 0 gypsy mdg 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ?-HP1, Mock ?-HP1, Piwi KD Figure 2-10. Depletion of Piwi from ovarian somatic follicle cells does not affect HP1 recruitment to piRNA clusters. (A) Western blotting of Piwi, HP1 and Lamin in OSC cells that were either mock treated (left lane) or treated with siRNA directed against piwi (right lane, Piwi KD). ChIP at flam (B, D) and 80EF (C, E) piRNA clusters in mock treated and Piwi KD OSC cells with antibodies specific to HP1 (B-C) or Piwi (D-E). 51 Loss of piRNA production from a single cluster results in global HP1 mislocalization We next sought to determine whether loss of piRNA production at a single piRNA cluster would affect HP1 recruitment to chromatin. Previous studies have shown that mutation of various RNA silencing components results in global mislocalization of HP1 on polytene chromosomes (Pal-Bhadra et al. 2004; Fagegaltier et al. 2009). Mutation of flam has been previously shown to result in loss of piRNA production (Brennecke et al. 2007) a nd upregulation of the gypsy retroelement (Prud'homme et al. 1995). In order to obtain a genome-wide view of HP1 chromatin association in flam mutants, we examined the localization of HP1 to highly replicated salivary gland polytene chromosomes from either wild type or flam1 mutant third instar larvae by indirect immunofluorescence using ?-HP1 antibodies. In wild type, HP1 localizes predominantly to a concentration of heterochromatin where the centromeres of each chromosome coalesce, termed the chromocenter (Figure 2-11A, green). In contrast, flam1 mutants display expansion of HP1 at the chromocenter. Spreading of HP1 is apparent on the second and third chromosomes, but not on the X chromosome, where flam is located. As a reference, we also examined the localization of the chromatin insulator protein Mod(mdg4)2.2, which is unchanged in localization between wild type and flam1 (Figure 2-11A, red). The extent of HP1 chromocenter expansion is comparable to the level of HP1 expansion that we observe in spn-EhlsE1/spn-EhlsE616 mutants (Figure 2-12). A lesser degree of HP1 expansion was also observed in flamBG02658/ flamKG00476 mutants (data not shown). Finally, total HP1 levels are unchanged in flam1 whole flies compared to wild 52 type (Figure 2-8). These results indicate a global change in HP1 localization resulting from inactivation of a single piRNA cluster. We reasoned that accumulation of HP1 at the chromocenter of flam1 mutants may result in an increase in silencing at pericentromeric sites. Therefore, the expression of transcriptional reporters at 42AB or 80EF piRNA clusters, which are located on different chromosomes from the flam locus, was examined in flam1 mutants. Compared to wild type, flam1 mutants harboring a P element insertion at either 42AB or 80EF piRNA clusters display mildly decreased pigmentation suggesting increased silencing at these distinct pericentromeric loci (Figure 2-11B). 53 Figure 2-11. Mutation of the flam piRNA cluster results in global HP1 redistribution. (A) Wild type (left) and flam1 (right) polytene chromosomes stained with antibodies directed against HP1 (green) and a reference protein Mod(mdg4)2.2 (red). DNA is stained with DAPI (blue). Chromosome arms are labeled, and insets of the enlarged chromocenter are shown. (B) Adult eyes of wild type and flam1 mutants carrying a mini- white transgene inserted in 42AB (top row) and 80EF (bottom) piRNA clusters. (C) Degree of eye pigmentation due to expression of the DX1 transgene array at 50C on chromosome 2L, which undergoes repeat-induced heterochromatic silencing, in wildtype, flam1/+, and flam1 female flies and wildtype and flam1 male flies. Scoring of variegation in the eye is categorized into five groups that range between light (few pigmented facets) to dark (almost all pigmented facets). Percentage of flies falling into each category was graphed. Representative eyes are shown on right. 54 WT X 3L 3R 2R 2L 4 HP1 Mod(mdg4)2.2 HP1 Mod(mdg4)2.2 HP1 Mod(mdg4)2.2 HP1 Mod(mdg4)2.2 X 2R 3R 2L 3L 4 WT fla m 1 /+ fla m 1 WT fla m 1 Females Males 0 10 20 30 40 50 60 Light (L) Medium-Light (ML) Medium (M) Medium-Dark (MD) Dark (D) Percentage of flies score d L ML M MD D A B Merged Merged flam1 WT 42AB 80E F flam1 C 55 WT spn-E hls-E1 /spn-E hls-E616 HP1 Mod(mdg4)2.2HP1 Mod(mdg4)2.2 HP1 Mod(mdg4)2.2 Merged Merged HP1 Mod(mdg4)2.2 Figure 2-12. spn-E hls-E1 /spn-E hls-E616 mutants display accumulation of HP1 at the chromocenter. Wild type and spn-Ehls-E1/spn-Ehls-E616 polytene chromosomes stained with C1A9 antibody directed against HP1 (green) or a reference protein Mod(mdg4)2.2 (red). DNA is stained with DAPI (blue). 56 Mutation of the flam piRNA cluster suppresses heterochromatic silencing at a distant site Finally, to verify HP1 genome-wide redistribution in flam1mutants, we examined the effect of flam1 on the silencing of a centromere distal heterochromatic site on a different chromosome. The DX1 transgene array consists of seven mini-white P elements with one inverted copy at a normally euchromatic site at 50C on chromosome 2R (Dorer and Henikoff 1994). Due to this configuration, the array forms ectopic repeat induced heterochromatin and displays a variegated phenotype similar to PEV that is dependent on HP1. Expression of the DX1 array was assessed based on variegation of eye pigmentation in wild type, heterozygous flam1/+, and homozygous or hemizygous flam1 mutants (Figure 2-11C). Due to a wide range of eye coloration, variegation was scored by categorization into five groups that ranged between Light (few pigmented facets) to Dark (almost all facets pigmented). For females, 3% of wild type was classified as Dark, while 29% of flam1/+ and 52% of flam1 mutants displayed the same high level of pigmentation. In males, 15% of wild type was scored as Medium-Dark or Dark while 40% of flam 1 males fell into these categories. These results indicate that mutation of flam can suppress heterochromatic silencing in trans. Taken together with the HP1 centromeric expansion in polytene chromosomes and increased pericentromeric silencing in flam1 mutants, there appears to be a global redistribution of HP1 resulting from the loss of piRNA production from a single locus. 57 DISCUSSION In this study, we tested directly whether the Argonautes AGO2 or Piwi recruit HP1 to chromatin. As candidate sites for Argonaute/HP1 interaction, we examined whether piRNA clusters may be heterochromatic using both genetic and molecular approaches. First, P elements inserted at or near pericentromeric piRNA clusters were assayed as transcriptional reporters, and these transgenes were found to display variegated expression that is increased in heterochromatin mutants. Next, ChIP with ?- HP1 antibodies showed that HP1 associates with piRNA clusters at levels significantly above euchromatic sites. However, mutation of piwi, aub, or AGO2 leads to a modest increase in silencing of transcriptional reporters as well as an increase of HP1 association at piRNA clusters in heads. In ovarian somatic follicle cells, in which both Piwi and HP1 are highly expressed, depletion of Piwi results in little or no change in HP1 recruitment to piRNA clusters and TEs. Furthermore, loss of piRNA production at a single locus results in expansion of HP1 at the centromere. In these flam1 mutants, silencing of a distant heterochromatic transgene array is reduced, further indicating a global redistribution of HP1 and suggesting indirect effects. Taken together, the results argue against direct recruitment of HP1 or maintenance of its association by AGO2 or Piwi in the soma. AGO2 and Piwi are not required for HP1 association at piRNA clusters Several reasons dictated the choice of piRNA clusters as the focus of our analyses. First, both endo-siRNAs and piRNAs are generated from these loci (Brennecke 58 et al. 2007; Yin and Lin 2007; Chung et al. 2008; Czech et al. 2008; Ghildiyal et al. 2008; Kawamura et al. 2008). Next, we reasoned that at least some piRNA clusters are likely to be heterochromatic because of their strong bias toward TE-rich pericentromeric positions in the genome (Brennecke et al. 2007; Yin a nd Lin 2007), in close proximity to the vast majority of HP1 localization. In fact, early cloning attempts determined that the flam locus is located in a repetitive, TE rich heterochromatic region (Robert et al. 2001). Furthermore, the pericentromeric position of these clusters likely coincides with the transition between euchromatin and heterochromatin, corresponding to the borders of HP1 spreading. This characteristic allows variegation assays, which monitor the variable spreading of HP1 and heterochromatin, to be extremely sensitive. ChIP assays at the borders of HP1 spreading would also likely be optimally sensitive to both local and overall changes in HP1 chromatin association. Finally, piRNA clusters contain enough unique sequence for specific primer design and monitoring by directed ChIP analysis. Given that AGO2 is the predominant Argonaute expressed outside the gonad that participates in the silencing of TEs in the soma, we tested whether AGO2 could recruit HP1 to chromatin in somatic tissue. Moreover, it has been shown that AGO2 mutants exhibit mislocalization of HP1 (Deshpande et al. 2005; Fagega ltier et al. 2009). However, our results show that mutation of AGO2 results in a strong increase of silencing of transcriptional reporters at or near piRNA clusters and a mild increase of HP1 chromatin association in heads. Given the extent of increased silencing in the AGO2 mutant compared to piwi or aub mutants, which accumulate HP1 on chromatin to a similar degree, a posttranscriptional step of silencing likely contributes to the negative effects observed on transcriptional reporters. AGO2 mutants show a plethora of cellular 59 defects during early nuclear divisions but develop normally and are fertile suggesting that effects on these various processes as well as HP1 localization are mild or otherwise compensated for (Deshpande et al. 2005). Therefore, AGO2 is unlikely to be required for HP1 recruitment in this tissue. Additionally, we find that HP1 association at piRNA clusters does not depend on the presence of Piwi. Our analysis of piRNA clusters included flam, a primary piRNA cluster, and 80EF, a germline piRNA producing locus. We examined both flam and 80EF clusters in somatic head tissue and ovaries, which are a mixed population of somatic follicle and germline derived cells. In heads, there is no apparent requirement for piwi with respect to HP1 recruitment to the piRNA clusters or to TEs that were examined. In our study, Piwi chromatin association was detected only in OSC cells, and its presence is dispensable for HP1 chromatin association. The flam piRNA cluster produces high levels of primary piRNA in OSC while 80EF is active for piRNA production in germ cells but not in OSC (Li et al. 2009a; Malone et al. 2009; Saito et al. 2009). Nonetheless, Piwi associates with both the flam and 80EF clusters at comparable levels, suggesting that the amount of piRNA production from a particular locus does not correlate with Piwi chromatin association. Furthermore, the pattern of Piwi chromatin association in OSC differs from that of HP1 in that there is no particular enrichment of Piwi at TEs above euchromatic sites and only a minor accumulation at a few sites in the flam and 80EF piRNA clusters. When Piwi levels were reduced by siRNA knockdown, Piwi chromatin association was essentially abolished but HP1 recruitment was not affected except for a two-fold decrease over the 1360 element. Previous studies suggested that the 1360 element may be responsible fo r nucleating heterochromatin on 60 the largely heterochromatic fourth chromosome and further showed that mutation of factors representing all RNA silencing pathways, piwi, aub, spn-E, Dcr-1, and Dcr-2, affect 1360 dependent heterochromatic silencing (S un et al. 2004; Haynes et al. 2006). Unlike the results in adult heads, no accumulation of HP1 over piRNA clusters was detected as a result of Piwi knockdown in OSC cells. This discrepancy may reflect differential effects in distinct cell types or the length of the Piwi knockdown in OSC cells, which was at least adequate to essentially eliminate Piwi chromatin association. In a related but independently derived ovarian somatic follicle cell line (OSS), Piwi and HP1 do not colocalize in the nucleus (Lau et al. 2009), and this finding supports the conclusion that Piwi does not direct HP1 recruitment in this cell type. Also consistent with our results, HP1 remains localized to the chromocenter in salivary gland polytene chromosomes in piwi null mutants (Pal-Bhadra et al. 2004; Brower-Toland et al. 2007). We conclude that association of HP1 with chromatin can occur independently of AGO2 and piwi in somatic tissue. A previous study addressed the role of the germline piRNA pathway in HP1 association with transposable elements. The spn-E gene controls predominantly germline piRNA production but does not affect the somatic piRNA pathway (Malone et al. 2009). ChIP was used to show that spn-E mutants display significantly decreased levels of H3K9me3 and HP1 at telomeric Het-A but similar to wildtype HP1 levels at the I-element and copia TEs, which are distributed throughout the genome (Klenov et al. 2007). This modest reduction of HP1 at Het-A was apparent in ovaries bu t not in carcasses, which contain only somatic tissue. One caveat to this study is that ChIP was performed using primers that detect all TEs matching a particular sequence, thus measuring average HP1 61 and H3K9me levels on TEs across the genome. Nonetheless, this work suggests a limited role for the germline piRNA pathway in HP1 recruitment at the telomere. Additional candidate platforms for Piwi-dependent HP1 recruitment Several studies have shown that Piwi associates with at least some heterochromatic sites in the genome, but direct evidence that any of these sites serve as recruitment platforms for HP1 and subsequent spreading is lacking. The best characterized Piwi-associated site is the heterochromatic 3R-TAS subtelomeric region, which generates the abundant Piwi bound 20nt 3R-TAS piRNA. Surprisingly, the role of piwi at this location is transcriptional activation, as piwi mutants display increased transcriptional silencing of a nearby reporter transgene as well as an increase of HP1 association at 3R-TAS (Yin and Lin 2007). Likewise, we observe a mild corresponding increase in HP1 association and silencing at piRNA clusters in piwi mutants suggesting that piwi function could in fact oppose HP1 recruitment at multiple sites in the genome. Our results are consistent with the possibility that piRNA clusters act as boundaries to the spread of pericentromeric heterochromatin. The mechanism of Piwi dependent transcriptional activation has not been determined, but considering that Piwi interacts with the chromoshadow domain of HP1 (Brower-Toland et al. 2007), Piwi may compete for binding with other HP1 interactors such as Su(var)3-9 that promote heterochromatic silencing. Functions for piwi outside of the gonad 62 The majority of Piwi protein is found in both somatic and germline tissues of the gonad, yet piwi clearly exerts an effect on non-gonadal somatic tissues as well. RT-PCR analysis shows that the piwi transcript is readily detectable outside the gonad and in somatic cell lines (Rehwinkel et al. 2006; Brower-Toland et al. 2007), but the Piwi protein is difficult to detect (Brower-Toland et al. 2007). Nevertheless, mutation of piwi suggests important functions for this gene outside of the gonad. For example, piwi is essential for viability, and loss-of-function mutants display a variety of phenotypes manifest in various non-gonadal somatic tissues such as demonstrated in this study and others, which show a requirement for piwi in pairing-dependent silencing, nucleolar integrity, and chromatin insulator function (Pal-Bhadra et al. 2002; Grimaud et al. 2006; Lei and Corces 2006; Peng and Karpen 2007). For each of these chromatin related studies, it remains a possibility that even a small amount of maternally deposited Piwi could trigger early events in the oocyte or embryo that persist throughout development, manifesting phenotypes visible in adult somatic tissues. HP1 redistribution in piRNA pathway mutants Our results along with previous studies have demonstrated that HP1 mislocalizes from the chromocenter in a subset of piRNA pathway mutants. We found that polytene chromosomes of flam1 mutants exhibit expanded HP1 chromocenter distribution. This result is intriguing because the flam1 mutation affects a single piRNA cluster on the X chromosome but HP1 spreading to other chromosomes is apparent. A previous study 63 detected spreading of HP1 to euchromatic arms especially in spn-E mutants (Pal-Bhadra et al. 2004), and we confirmed this result albeit to a lesser degree, with spreading being comparable to the extent seen in flam1 mutants. Perhaps the increase of TE expression in RNA silencing mutants can stimulate HP1 recruitment and spreading from the centromere, which contains the highest concentration of TEs. In fact, transcription of pericentromeric repeats stimulates RNAi-dependent heterochromatin formation in fission yeast (Zofall and Grewal 2006; Chen et al. 2008; Kloc and Martienssen 2008). Redistribution of HP1 in RNA silencing mutants may indirectly affect silencing at various heterochromatic locations in the genome. Seemingly inconsistent with HP1 spreading, spn-E, aub, and piwi mutants display decreased silencing of P element transgene arrays such as DX1 and single insertions at pericentromeric regions on chromosomes 2 and 4 (Pal-Bhadra et al. 2004). In our study, we found that mutation of flam also results in loss of silencing at DX1, which is distant from the flam locus. This reduced silencing in trans could not be due to posttranscriptional events as there are no shared sequences between DX1 and the flam locus. Therefore, we consider the possibility that there exists a finite pool of HP1 that accumulates at the centromere in flam and other RNA silencing mutants at the cost of reduced density and reduced silencing at other heterochromatic regions such as the transgene array, the fourth chromosome, and the telomere. The concept of a limited population of HP1 was suggested previously to explain the finding that the Y chromosome behaves as a suppressor of variegation by acting as a sink for HP1 (Dorer and Henikoff 1994). Conclusions 64 Studies in multiple organisms have identified or suggested alternative mechanisms to RNA silencing for the recruitment of HP1 to chromatin. In fission yeast, overlapping and redundant RNAi-dependent and independent mechanisms of heterochromatin formation have been elucidated. In mouse cells, HP1 localization to pericentromeric heterochromatin was found to be RNase A sensitive suggesting that an RNA moiety may be involved in HP1 recruitment (Maison et al. 2002). Our data indicate that heterochromatin can form independently of RNA silencing in Drosophila. It will be interesting to determine if any of these alternative mechanisms of heterochromatin formation are conserved throughout evolution. 65 ACKNOWLEDGEMENTS I would like to thank Ryan Dale for computational support and the generation of Figure 2-1 and C. Berg, A. Beyer, J. Birchler, V. Corces, J. Eissenberg, S. Elgin, G. Hannon, S. Henikoff, H. Lin, P. Macdonald, U. Sch?fer, P. Schedl, and M. Siomi for fly strains and antibodies. I am indebted to K. Saito and M. Siomi for OSC cells and detailed culture protocols, Y. Zhang for ChIP protocols, and members of the Lei laboratory for discussions. 66 AUTHOR CONTRIBUTIONS All the experiments were performed by N.M., and were designed by N.M. and E.P.L. E.P.L. contributed to the writing of the manuscript presented in this chapter. 67 MATERIALS AND METHODS Drosophila strains Fly stocks were maintained at 25?C on standard cornmeal medium. Lines containing P{EPgy2}DIP1 EY02625 and P{EPgy2}EY08366 were obtained from the Bloomington Drosophila Stock Center, and a line harboring PBac(PB) c06482 was obtained from the Exelixis Collection at Harvard Medical School. Genomic coordinates of these P-element insertions were confirmed by PCR with primers specific to the P- elements and flanking genomic sequences followed by sequencing. For transcriptional reporter assays, transgenes were crossed or recombined into mutant backgrounds and scored against crosses to yw67c23 as a reference. For ChIP and immunofluorescence, Oregon-R was used as a wildtype control. The y v f mal flam1/FM3 stock was selected for heterozygous females each generation to prevent mobilization and accumulation of TEs. For the DX1 variegation assay, DX1/CyO was crossed to y w v f mal flam1/FM7c; CyO/Sp flies or yw67c23 ; CyO/Sp as a reference. Transcriptional reporter and eye pigmentation assays Eye pigmentation of 40 to 60 adult males six days of age was examined, and representative eye photos were taken. To quantify overall levels of eye pigmentation, the heads of 25 male flies of each genotype were dissected, and eye pigmentation was measured as previously described (Pal-Bhadra et al. 2004). Briefly, heads were 68 homogenized in 0.8 ml of methanol, acidi fied with 0.1% HCl and centrifuged. The absorbance of the supernatant was measured at 480 nm. Chromatin immunoprecipitation Crosslinking and sonication Wildtype (Oregon-R) heads or ovaries were dissected on dry ice. Fly heads/ovaries were washed in 5 ml PB S containing 0.01% Triton X-100 and centrifuged for 1 min at 500 rcf to pellet heads. Supern atant was discarded, and 1 ml of crosslinking solution (50 mM HEPES, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, and 1.8% formaldehyde) and 3 mL n ?heptane were added. The mixture was shaken vigorously for 20 min at room temperature. Supernatant was discarded, and heads were resuspended in 5 ml PBS containing 125 mM glycine and 0.01% Triton X-100. The mixture was shaken for 5 min at room temp erature. Supernatant was discarded after centrifugation, and 5 ml of ice cold PBS containing 0.01% Triton X-100 was added. Supernatant was removed and heads were resuspended in 5 ml ice cold PBS containing 0.01%Triton X-100 and protease inhibitors (R oche). Heads were Dounce homogenized with pestle A (Kontes) for tissue disaggregation and complete homogenization. The mixture was centrifuged at 400 rcf for 1 min, and supernatant was transferred to a fresh tube. Supernatant was centrifuged at 9190 rcf for 5 min at 4?C and supernatant discarded afterwards. The pellet was resuspended in 5 ml ice cold Cell Lysis Buffer (5 mM PIPES, pH 8, 85 mM potassium chloride, 0.5% Nonidet P40 (NP40) and protease inhibitors). 69 The mixture was Dounce homogenized with pestle B to release the cell nuclei and centrifuged at 9190 rcf for 5 min at 4?C. S upernatant was removed, and the pellet was resuspended in 1 ml of ice cold Nuclear Lysis Buffer (50 mM Tris HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease inhi bitors) and incubated for 20 min at 4?C. Then 0.5 ml ice cold IP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA pH 8.0, 16.7 mM Tris.HCl, pH 8.0, 167 mM Na Cl and protease inhibitors) and 0.3 g of acid washed 212?300 micron glass beads (Sigma) was added. The mixture was sonicated in ice water 8 times for 30 s with 30 s intervals, transfe rred to microfuge tubes and centrifuged at 18407 rcf for 10 min at 4?C. Quality control of input chromatin 100 ?l of chromatin was adjusted to 200 ?l with IP dilution buffer and decrosslinked at 65 oC overnight. 2?l of proteinase K at 20 mg/ml (Invitrogen) was added, and the mixture was incubated at 55 oC for 2 h. Chromatin was extracted twice with equal volume of phenol:chloroform (Sigma) and once with chloroform (Sigma). 2 ?l of glycogen and one-tenth volume of 3M NaOAc, pH5.2 and 2.5 volumes of ethanol were added. The mixture was incubated for 30 min at -20?C, centrifuged and wa shed twice with 70% et hanol. The pellet was dissolved in 50 ?l water. Chromatin size was checked on a 1% agarose gel. DNA was quantified using Nanodrop ND1000 spectrophotometer. Preparation of Protein A beads 70 rProtein A agarose beads (GE Healthcare) were washed with IP dilution buffer 3 times and blocked in IP dilution buffer containing 1% BSA rotating at 4?C overnight. Beads were washed three times in IP dilution buffer and resuspended in IP dilution buffer for later usage. Chromatin preclear Chromatin was diluted three to five times with IP dilution buffer (depending on the DNA concentration). 30 ?l of washed Protein A agarose beads were added to each fraction of diluted chromatin to be used in later immunoprecipitation and incubated at 4?C for 2 h. Immunoprecipitation Same volume of precleared chromatin was aliqouted for each IP sample. 3-5 ?l of antibodies were added to aliquoted chromatin and incubated rotating at 4?C overnight. 30 ? l of washed beads previously blocked with IP dilution buffer containing 1% BSA were added to each IP sample. The mixture was incubated at 4?C for 4 h. Chromatin-bound beads were centrifuged at 587 rcf for 2 min, and supernatant was discarde d. Beads were washed three times with 1 mL Low salt wash buffer (0.1% SDS, 1.0% Trit on X-100, 2.0 mM EDTA pH 8.0, 20 mM Tris.HCl, pH 8.0, 150 mM NaCl), three times with High salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2.0 mM EDTA, pH 8.0, 20 mM Tris .HCl, pH 8.0, 500 mM NaCl), and two times with LiCl buffer (0.25 M LiCl, 1 mM EDTA, pH 8.0, 10 mM Tris HCl, pH 8.0, 1% NP40, 1% SDC) for 5 min with rotation at RT and pelleted at 587 rcf for 2 min. Chromatin was eluted with 200 ?l of freshly prepared IP Elution Buffer (0.1 M NaHCO3, 1% SDS) at 65?C for 30 min. This step was repeated one more time. NaCl (5 M, 20 ?l), EDTA (0.5 M, 8 ?l), Tris (1 M, pH 8.0, 16 ?l) were added to 400 ?l of eluted chromatin and incubated overnight at 65 oC. 4 ?l of Proteinase K at 20 71 mg/ml were added for 3 h at 55 oC. Chromatin was extracted with phenol:chloroform and precipitated as described before. IP samples were dissolved in 50 ?l water. ChIP quantification The quantities of target genomic regions precipitated by different antibodies were calculated as percent input based on four-point standard curves constructed from input DNA for each primer set. Standard deviation of each PCR performed in quadruplicate was calculated to determine the error of measurement. Two independent ChIP samples were analyzed, and similar results were obtained. ChIP primers were designed to be unique, detecting only sequences present in the flam and 80EF piRNA loci and verified by in silico PCR. All primers (Table 2-2 and 2-3) were checked for both specificity and efficiency by standard agarose gel electrophoresis and real time PCR respectively. Primers to piRNA clusters amplify in the same DNA dilution range as primers specific to hsp26 and yellow single copy genes compared to high copy TE elements (Figure 2-13). Culture of OSC cell line and siRNA knockdowns The OSC line was maintained and Piwi siRNA knockdown was performed as previously described (Saito et al. 2009). Briefly, 3 x 106 trypsinized cells were resuspended in 0.1 mL of Solution V of th e Cell Line Nucleofector Kit V (Amaxa Biosystems) and mixed with 200 pmol of siRNA duplex. Transfection was conducted according to the manufacturer?s protocol using the nucleofector program T-029, and the transfected cells were incubated at 25?C for 48 hrs. Protein knockdowns were verified by 72 Western blotting, and ChIP assays were performed on mock and piwi siRNA transfected cells (5x10 6 cells per IP). Immunostaining of polytene chromosomes Preparation and immunostaining of salivary gland polytene chromosomes was performed as described previously (Gerasimova et al. 2000). Primary antibodies directed against HP1 (Covance) and Mod(mdg4)2.2 (generated similarly as in Mongelard et al. 2002) and Alexa Fluor 488 labeled anti-guinea pig or Alexa Fluor 594 labeled anti-rabbit secondary antibodies (Invitrogen-Molecular Probes) were used. The chromosomes were viewed using a Leica epifluorescence microscope and photographed using a Hamamatsu digital camera. DX1 variegation assay Eye pigmentation of 100 to 200 flies was scored. The scoring of variegation was categorized into five groups: Light, Medium-Light, Medium, Medium-Dark and Dark corresponding to the percentage of pigmented facets. Percentage of flies falling into each category was graphed. Representative eye photos were taken. 73 20 21 22 23 24 25 26 27 28 29 30 25 12.5 6.25 3.125 DNA [ng/ul] C t value hsp26 yellow 1360 TART 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 21 22 23 24 25 26 27 28 29 30 25 12.5 6.25 3.125 DNA [ng/ul] C t value hsp26 yellow 1360 TART A B C D E F G H I J K L M N O P flamenco primers 80EF primers A B Figure 2-13. ChIP primer efficiency and specificity. PCR amplification efficiency and specificity of ChIP primers at (A) flam and (B) 80EF piRNA loci are graphed as a function of cycle threshold (Ct) values over DNA concentration. Ct values of standard curves of input from a representative experiment were graphed to show that primers to piRNA clusters amplify in the same DNA dilution range as primers specific to single copy genes hsp26 and yellow compared to high copy TE elements. 74 Table 2-2. Primer set sequences used for ChIP at the flam piRNA cluster. Sequence 5? to 3? Genomic coordinates 1 cgttcatgtcgttccacaac chrX:21473147+21473333 187bp tgcacggatcgtggttatta 2 aaaccacttcgcggatttc chrX:21495641+21495810 170bp tgcattttgatttcttgtgctc 3 aacgaggccagattcaacat chrX:21497545+21497751 207bp gaatcagtacgagggcaagg 4 caagttggggtttcgtgttt chrX:21504581+21504730 150bp attgaaccttaccccgacaa 5 ggagtgggatggatagacga chrX:21510544+21510729 186bp cctggacacaggaccaaagt 6 ctcgggattttgcgttacat chrX:21526571+21527094 231bp ggcagctaaccgtggataaa 7 gtggcttcacaaaacacgac chrX:21527157+21527376 220bp cgaaggcttacacgcaagat 8 cctaccaacccagcgaataa chrX:21537802+21538037 236bp tgctcttaagcctgcgaaat 9 cgatccgtttatgcaggtct chrX:21539214+21539437 224bp ctgccaacaaatccatttcc 10 tgcctgtcgtactttgcttg chrX:21543255+21543448 194bp ccaatgaattgccgctagtt 11 cgcgactgattggaagaact chrX:21586797+21586976 180bp tctaagcccaacgtacacga 12 tcaggattcctccagaggtg chrX:21604099+21604347 249bp ggccgctatgagtttcatgt 13 tgcgtgacgtaagcaaactc chrX:21605733+21605922 190bp ttttatcggtggtgggaaag 14 cgggtgtaggtcacttggtt chrX:21611603+21611784 182bp cagttaccaacgcaatcacg 15 tgcgtgccttttaaggagtc chrX:21618001+21618210 210bp cgctgaatgcgatagtgaca 75 Table 2-3. Primer set sequences used for ChIP at the 80EF piRNA cluster. Sequence 5? to 3? Genomic coordinates A aggcacatggatgaacaaca chr3L:23255817+23255966 150bp gtttggttaacgggcaacat B accgtgcatcccaatatcat chr3L:23257001+23257188 188bp ccaccaaaagaaagaacacg C aggacacacatgcttgctttt chr3L:23262518+23262618 101bp cgataaatcttcttttggcaga D tagcattacggcgaatggac chr3L:23271313+23271532 220bp ctctgcaataaagcgcacac E gcttcgaagaagtgcaatca chr3L:23277432+23277632 201bp ttttgagcgggttttattcg F ggacggtttgtttgtcttcg chr3L:23278084+23278273 190bp gactcgatgtggccatgata G ttttgcatgtggcaataatca chr3L:23281146+23281329 184bp cgcatcggatattgtctgtg H cgaggcatgtcgtagctgta chr3L:23290484+23290709 226bp gccctagtggcctcttctct I cctcattttcgcctcgatta chr3L:23291884+23292130 247bp aaaagaaccgcaagagagca J tcgatgagcaagatgtgagg chr3L:23295139+23295322 184bp aaacgagatggccaacaaag K agggtccggttctcttctgt chr3L:23300821+23301000 180bp aaaacttggttgccctgatg L tcgtggtgcagttgagagtc chr3L:23307902+23308093 192bp aagagcggcagagagtcaag M aaatcaaacggagtttctgtttct chr3L:23308478+23308657 180bp caagctcaaagtgccatcaa N tttcggaagctggtacaaag chr3L:23312351+23312521 171bp cgccgcttatattttgaacg O ctagtttttcagcgtgcttgg chr3L:23322270+23322429 160bp ctaagaaggcaattgcgaaag P ggagctattggagccgtcta chr3L:23332664+23332763 100bp tgtactcttgccatggttcg 76 CHAPTER 3 RNAi-INDEPENDENT ROLE FOR ARGONAUTE2 IN CTCF/CP190 CHROMATIN INSULATOR FUNCTION ABSTRACT A major role of the RNAi pathway in S. pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by ChIP-seq in two different embryonic cell lines was performed revealing that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. I propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. 77 INTRODUCTION RNA silencing is an evolutionary conserved mechanism that involves small RNAs bound to an Argonaute (AGO) protein that act as transcriptional or post- transcriptional regulators of gene expression. The paradigm for how RNA silencing controls gene expression at the chromatin level comes from studies in fission yeast, in which the RNA interference (RNAi) machinery establishes heterochromatin at the centromere and mating type locus to ensure proper chromosome segregation and to promote stability of repetitive regions. At the centromere, RNAs transcribed from pericentromeric repeats are processed by the Dcr1 endonuclease and Ago1 Argonaute protein, which leads to the recruitment of the histone H3K9 methyltransferase and Swi6/HP1 binding (reviewed in (Grewal and Elgin 2007)). In Drosophila, it remains unclear whether the RNAi pathway is involved directly in heterochromatin formation. The primary endogenous function of the RNAi/siRNA pathway is to silence the expression of transposable elements (TEs) in the soma (reviewed in (Okamura and Lai 2008)). Silencing is achieved by Dcr-2-mediated cleavage of double-stranded RNAs (dsRNAs) into 21-22 nt siRNA that are loaded into AGO2, which cleaves the target TE mRNA using its Slicer activity. Less well understood is the function of non-TE endo-siRNAs also produced by Dcr-2 activity and loaded into AGO2, which are generated from hairpin transcripts and regions of 3? overlap of convergent transcripts (3? cis-NATs). Two studies implicated AGO2 in heterochromatin formation based on mislocalization of HP1 and desilencing of pericentromeric transcriptional reporters in AGO2 mutants (Deshpande et al. 2005; 78 Fagegaltier et al. 2009). However, direct analysis of HP1 recruitment by chromatin immunoprecipitation (ChIP) and HP1-dependent silencing at small RNA generating loci led to the suggestion that AGO2 and other Argonaute genes may not be required for heterochromatin formation in the soma (Moshkovich and Lei 2010). Chromatin insulators are DNA-protein complexes defined functionally either as barriers that prevent the spread of silent chromatin or enhancer blockers that constrain enhancer-promoter communication. Unlike vertebrates, which possess only one known insulator protein, CTCF (reviewed in (Phillips and Corces 2009)), Drosophila employs at least five different insulator complexes. Two well-characterized insulators are the gypsy (also known as Su(Hw)) insulator and the Fab-8 insulator of the Abd-B locus in the bithorax complex (BX-C) (reviewed in (Bushey et al. 2008)). The gypsy and Fab-8 insulators harbor binding sites for the zinc-finger DNA-binding proteins Su(Hw) and CTCF, respectively, and both insulator complexes share a common component, CP190. Genome-wide insulator proteins are present at thousands of distinct DNA-binding sites but in diploid cells they concentrate at a small number of nuclear foci termed insulator bodies, which are dependent on CP190 for their integrity. Highly correlated at least with gypsy insulator function, insulator bodies have been proposed to serve as tethering sites for large chromosomal loops or other higher order chromatin structures. It has become increasingly apparent that DNA topology is a critical determinant of gene regulation. While enhancers activate their target promoters over long distances, insulators act to restrict these communications (reviewed in (Wallace and Felsenfeld 2007)). Insulators and other cis-regulatory regions in the Abd-B locus engage in numerous interactions, and the precise topology of the locus has been postulated to be a 79 central mechanism of tissue-specific Abd-B regulation (Cleard et al. 2006; Lanzuolo et al. 2007; Kyrchanova et al. 2008; Ba ntignies et al. 2011). However, the mechanism by which chromosome looping is achieved at this locus has not been elucidated. Vertebrate CTCF has been shown to mediate chromosomal looping at several developmentally regulated loci jointly with cohesin (reviewed in (Merkenschlager 2010)), and a recent study reported that CTCF promotes promoter-enhancer interactions genome-wide. However, it is unknown whether Drosophila CTCF can promote looping. In Drosophila, AGO2 or other RNA silencing factors appear to play important roles in chromatin and nuclear organization, such as formation of Polycomb Group (PcG) repression bodies (Grimaud et al. 2006) and gypsy chromatin insulator bodies (Lei and Corces 2006). Furthermore, AGO proteins ha ve been detected in the nucleus and are thought to have a functional role in that compartment. Overall, these studies suggest novel mechanisms by which RNA silencing affects gene expression on the level of higher order chromatin organization. Here, I hypothesized that similarly to the gypsy insulator, RNA silencing can affect the Fab-8 insulator. A comprehensive genetic analysis of diverse RNA silencing mutants on Fab-8 insulator function revealed that AGO2 was the only RNA silencing component to exert an effect. In order to test whether AGO2 may have a function on chromatin, ChIP-seq analysis of AGO2 in two Drosophila cell lines was performed. Instead of repetitive sequence, AGO2 associates primarily with euchromatic sites, the majority of which correspond to chromatin insulators. Intriguingly, AGO2 chromatin association does not correspond to regions of the genome that produce endo-siRNAs. I demonstrate that AGO2, but not its catalytic activity, is required for CTCF/CP190- 80 dependent Fab-8 insulator function. Additionally, AGO2 interacts physically with CP190 and CTCF. Chromosome conformation capture (3C) experiments demonstrate that CTCF/CP190-dependent looping interactions may regulate AGO2 recruitment to chromatin. Also, depletion of AGO2 leads to a decrease in chromosomal looping and, thus, altered gene expression. Therefore, I propose a novel RNAi-independent role for AGO2 on chromatin to promote or stabilize insulator-dependent looping interactions to define transcriptional domains throughout the genome. 81 RESULTS AGO2 associates with euchromatin and not repetitive sequences In order to obtain high-resolution information about the genome-wide chromatin association profile of AGO2, ChIP-seq analysis of AGO2 in S2 and S3 Drosophila embryonic cell lines was performed. ChIP was carried out using a previously characterized monoclonal antibody, 9D6, capable of isolating AGO2 and associated small RNAs (Kawamura et al. 2008). Greater than 9M reads per input or IP sample were obtained, leading to the identification of 3367 AGO2 bound sites between both cell types using a 5% false discovery rate threshold w ith the MACS algorithm (Zhang et al. 2008). Approximately 86% of AGO2 sites in S2 overl ap with those found in S3 suggesting that AGO2 genome-wide localization is mainly consistent between cell types. Comparing the fraction of total reads mapping to repetitive sequences indicates no enrichment of repetitive sequences in the IP versus input (chi-square test, p < 2e -16 ); therefore, I conclude that AGO2 localizes predominantly to euchromatic regions. Strikingly, the majority of AGO2 sites overlap with known chromatin insulator sites throughout the genome. As a model region, I inspected the 300 kb BX-C Hox gene cluster and observed association of AGO2 with all known cis-regulatory domain boundaries in both cell lines (Figure 3-1). These insulators include the Abd-B locus boundary elements Mcp , Fab-6, Fab-7 , and Fab-8. We obtained a similar ChIP-seq profile with lower signal using an independent ?-AGO2 polyclonal antibody (Meyer et al. 2006). Moreover, three independent an tibodies capable of immunoprecipitating 82 AGO2 (Jiang et al. 2005; Meyer et al. 2006; C zech et al. 2008) show similar enrichment profiles at the Abd-B locus as determined by ChIP followed by quantitative PCR (data not shown; Moshkovich et al. 2011). For subsequent genome-wide binding site analyses, 9D6 data was used exclusively because of its high signal-to-noise ratio and well- characterized specificity (Kawamura et al. 2008) (Fi gure 3-1, data not shown). 83 Figure 3-1. ChIP-seq profiles of AGO2 in S2 and S3 cells at BX-C. AGO2 ChIP-seq profiles of input DNA and IPs in S2 and S3 cells compared with tiling array ChIP data for CTCF, CP190, GAF (Negre et al. 2010), Trx-N, Pho, and Pc (Schuettengruber et al. 2009) in indicated cell types or embryos over the BX-C region (top) and Abd-B locus (bottom). Coding sequences, promoters, and cis-regulatory regions are shown. ChIP-seq scales are in reads per million unique mapped reads. Input samples are shown on the same scale relative to respective IP and are therefore directly comparable. ChIP-chip data are expressed either as log2 (IP/input) or as MA2C score. The bottom of each scale bar indicates zero. AGO2 ChIP-seq analysis was performed by Ryan K. Dale. 84 CP190 (embryo) CTCF (S2) GAF (embryo) Pho (embryo) 9D6 AGO2 (S2) 9D6 AGO2 (S3) Pc (embryo) 12730000 12740000 12750000 12760000 12770000 12780000 12790000chr3R Abd-B RE Abd-B RC Abd-B RA Abd-B RD Abd-B RB Fab-7PRE iab-7enhFab-7 Fab-8PRE iab-8enhFab-8 12550000 12600000 12650000 12700000 12750000Abd-B abd-A abd-A abd-A CG10349Glut3CG31275 CG31275 Abd-B RE Abd-B RC Abd-B RA Abd-B RD Abd-B RB Fab-7 Fab-8Fab-6Mcp zoom CP190 (embryo) CTCF (S2) GAF (embryo) 9D6 AGO2 (S2) 9D6 AGO2 (S3) PREenhancer insulator 12 3 4 5 6 7 8 9 10 11 12 13Primers 14 iab-2PREbxdPREbxPRE Mueller AGO2 (S2) Mueller AGO2 (S2) 22 24 11 4 5 4.3 22 24 11 4 5 4.3 Trx-N (embryo) Trx-N (embryo) 17.3 13.7 12.5 3.2 22 15.7 12.5 4.6 7.7 4.7 scaled tag density scaled tag density scaled tag density MA2C score MA2C score MA2C score log2 (IP/input) log2 (IP/input) log2 (IP/input) log2 (IP/input) log2 (IP/input) log2 (IP/input) MA2C score scaled tag density scaled tag density scaled tag density 85 AGO2 colocalizes with chromatin insulator sites throughout the genome Consistent with binding at BX-C boundary sites, approximately 62% of AGO2 sites overlap with known chromatin insulator proteins. Comparison of AGO2 ChIP-seq profiles with previously determined genome-wide ChIP tiling array analyses indicates extensive overlap with the insulator proteins CP190, CTCF, BEAF-32, and modest similarity to Mod(mdg4)2.2 compared to random expectation (Figure 3-2 (A-B); Moshkovich et al. 2011). In contrast, AGO2 sites display no statistically significant overlap with the gypsy insulator protein Su(Hw), indicating specificity of the AGO2 correspondence with CTCF/CP190 insulators. In order to confirm the genome-wide colocalization of AGO2 with insulator proteins and specific association with euchromatin, highly replicated polytene chromosomes of third instar larvae were stained by indirect immunofluorescence. AGO2 staining is mainly observed at euchromatic DAPI interbands, which correspond to decondensed regions of the genome bearing the majority of transcribed genes (data not shown; Moshkovich et al. 2011). In contrast, AGO2 is not visible at the heterochromatic chromocenter, at which the centromere of each chromosome coalesces. In AGO251B null mutants (Xu et al. 2004), this staining pattern is dramatically reduced, verifying the specificity of the antibody (data not shown). In wild type, modest genome-wide colocalization is observed between AGO2 and CTCF while more extensive overlap is seen between AGO2 and CP190, consistent with the ChIP-seq results (data not shown). 86 Figure 3-2. Overlap between genome-wide binding sites of AGO2, insulator, TrxG/PcG, transcription related factors, and promoters. (A) Binary heat map of AGO2 binding sites ordered by supervised hierarchical clustering. Each column represents one of the 3367 AGO2 binding sites across both S2 and S3 cell types, and each row represents overlapping binding sites for a particular factor across all available data sets. A mark in a row indicates that the indicated protein colocalizes with AGO2 at that site. AGO2 sites are classified into functional groups (endo-siRNA, PcG, and insulators). Feature counts for each factor and the number of features that intersect with the set of all AGO2 sites are shown. Corresponding percentages of overlap for each factor or for AGO2 are represented as grayscale values (left). (B) Heat map of log2 enrichment scores for pairwise comparisons of binding sites for AGO2, CP190, CTCF, 3? cis-NATs, and endo-siRNA clusters with additional data sets. Enrichment score was calculated by dividing the actual overlapping feature count by the median overlapping feature count from 1000 random shufflings of features. Empirical p values reported in the text are the percentile of the actual overlapping feature count in this null distribution. Color scale corresponding to enrichment value is indicated (right). Positive values indicate significant enrichment while negative values indicate significant negative correlation of enrichment. Self-self comparisons are indicated in grey, and pairwise comparisons that are not statistically significant (p > 0.001) are indicated in white. Numbers along top of each column indicate the total number of features in each data set, and the number of sites that interact with all AGO2 sites are indicated in parentheses. 87 (C) Half of AGO2 binding sites correspond to promoters. Profile of S2 AGO2 ChIP-seq tag density subtracted by input density around transcription start sites (blue) or termination sites (red) from coding genes (FlyBase R5.23) generated using CEAS. (D) AGO2 associates preferentially with active promoters. Profile of S2 AGO2 ChIP- seq tag density subtracted by input density around TSSs associated (orange) or not associated (green) with H3K4me2 and Pol II 250 bp upstream or 750 bp downstream. AGO2 binding site comparisons with other datasets was performed by Ryan K. Dale. 88 89 AGO2 associates with active promoters Like insulator proteins, over half of AGO2 binding sites are located at promoters. Extensive promoter association has been reported for the insulator proteins CP190, CTCF, Mod(mdg4)2.2, and BEAF-32 but not Su(Hw) (Bushey et al. 2009; Jiang et al. 2009; Smith et al. 2009), with a preference for active promoters (Negre et al. 2010). Genome-wide, 61% of AGO2 sites in S2 cells are found within 250 bp upstream of a transcription start site (TSS), with a slight bias upstream of the TSS (Figure 3-2C). In contrast, no enrichment of binding is seen proximal to transcription termination sites (as defined by the presence of polyA sites). Next, AGO2 binding sites at TSSs associated with RNA Pol II and H3K4me3 in the body of the gene were compared to those lacking these active marks of transcription revealing that AGO2 associates preferentially with active promoters (Figure 3-2D), corresponding to approximately 14% of all active promoters. Consistent with this finding, AGO2 associates with all five active Abd-B promoters in S3 cells but with only the RB and RE (also known as m and ? respectively) inactive promoters in S2 cells (Figure 3-1). Moreover, AGO2 associates with the iab-8 enhancer in S3 but not in S2 cells, suggesting that its chromatin association with certain enhancers and promoters may be dependent on active transcription. 90 AGO2 chromatin association does not correspond to regions of the genome that produce endo-siRNA In contrast to its association with insulator proteins, AGO2 genome-wide localization does not coincide with regions of the genome that produce Dicer-dependent endo-siRNA. First, the genome-wide distribution of AGO2 binding sites was compared to a set of 257 clusters of high-density AGO2-bound unique endo-siRNAs in S2 cells (Czech et al. 2008; Ghildiyal et al. 2008; Ka wamura et al. 2008; Okamura et al. 2008) demonstrating overlap with less than 1% of AGO2 sites, which is not statistically significant compared to random expectation (Figure 3-2 (A-B), p=0.35). As an additional test, the densities of unique endo-siRNA matching AGO2 chromatin binding sites in comparison to regions of the genome known to produce endo-siRNAs were calculated. Only thirty percent of 3? cis-natural antisense transcripts (cis-NATs) have been shown to produce Dicer-dependent endo-siRNAs (Okamura et al. 2008; Roy et al. 2010). The endo-siRNA densities of all known 3? cis-NATs were used to perform a conservative comparison to AGO2 chromatin-associated sites. Then, the endo-siRNA densities of sets of AGO2 binding regions and 3? cis-NATs shuffled throughout the genome in order to randomize their positions were calculated. Normalized to relative random expectations, substantially more 3? cis-NATs produce >8 endo-siRNAs per kb compared to AGO2 sites, which produce much lower levels of endo-siRNA (Figure 3-3 (A-B)). Production of such a low level of endo-siRNA at AGO2 sites may be due to the fact that AGO2 bound sites are associated with active transcription. These observations suggest that actively transcribed regions may produce more endo-siRNA than transcriptionally silent 91 regions. In fact, the top 5% highest endo-siRNA density AGO2 sites cluster with marks of active transcription (dat not shown, Moshkovich et al. 2011). Therefore, the endo- siRNA density analysis using Pol II and H3K27me3-bound regions, which represent transcriptionally active and inactive sites respectively, was repeated. Overall, Pol II- bound regions produce moderately higher levels of endo-siRNA than AGO2 sites while H3K27me3-bound regions produce lower levels of endo-siRNA compared to respective random expectations (Figure 3-3 (A-B)). Similar results were obtained using a nuclear library of S2 endo-siRNA (Figure 3-3 (C-D); Fagegaltier et al. 2009). These results suggest that regions of active transcription tend to produce low levels of endo-siRNA, and the majority of AGO2 binding sites correspond to little or no endo-siRNA production. 92 Figure 3-3. Distribution of endo-siRNA densitities for AGO2 binding sites compared to 3? cis -NATs and transcriptionally active or inactive regions. (A) Enrichment scores of endo-siRNA read densities in S2 AGO2 sites (black), 3' cis- NATs (Okamura et al. 2008) (red), H3K27me3 domains (Schuettengruber et al. 2009) (yellow), and S2 Pol II domains (Negre et al. 2010) (blue) expressed as a ratio of actual over random. For each bin, the enrichment score is calculated as (actual + 0.01) / (randomized + 0.01) to avoid dividing by zero. No enrichment with respect to random is indicated by a horizontal dashed line at 1. (B) Complementary cumulative distributions of the density of endo-siRNA reads in S2 AGO2 sites (black), 3' cis-NATs (red), H3K27me3 domains (yellow), and S2 Pol II domains (blue). Solid lines show actual distribution while dotted lines show the average distribution over 1000 random intrachromosomal shufflings of binding sites. (C) As in A, but using siRNAs bound to NLS-P19, a viral suppressor protein directed to the nucleus that acts upstream of AGO2 by binding double stranded siRNAs produced by Dcr-2 cleavage. (D) As in (B) but using NLS-P19 bound siRNAs. SiRNA analyses were performed by Ryan K. Dale. 93 c C P 1 9 0 ( e m bryo)TFS2GAh2DPe P60O C P 1 0 m P( 91 (0 P1m 1 ( P1 PC10 1C0mSb3yTAcb7A45 G8a1 h2D1Cm0O 9-ARFT)2GE h2DemeOi9n1eMb9 h2D10mCOpozAss h2DeP( O ur7 FRlM brtA 73 tFo h3Rtx3 zd7 3ryo MO CNC CN PNC PN 1NC 1N 9NC 9N 0NC 0N 2gI)pP6Ah2D1 9 PO C P 1 0 m P( 91 (0 P1m 1 ( P1 PC10 1C0mSb3yTAcb7A45 ur7 FRlM brtA 73 tFo h3Rtx3 zd7 3ryo MO G8a1 h2D1Cm0O 9-ARFT)2GE h2DemeOi9n1eMb9 h2D10mCOpozAss h2DeP( O CNCC CNC CNPC CNP CN1C CN1 CN9C 2gI)pP6Ah2D1 9 PO C P 1 0 m P( 91 (0 P1m 1 ( P1 PC10 1C0m/1C 0m Sb3yTAcb7A45 ?7 3Rt For Ao ?ATFtbTA?F tlA Mo7 bA 7b 3y T 3Rtx3zG8a1 h2D1Cm0O 73ryoMF?byG8a1 9-ARFT)2GE h2DemeO3Rtx3z 73ryoMF?by9-ARFT)2GE i9n1eMb9 h2D10mCO3Rtx3z i9n1eMb9 73ryoMF?by pozAss h2DeP( O3Rtx3z 73ryoMF?bypozAss h CNC CNP CN1 CN9 CN0 CN CN( bryo)TFS2GAh2DPe P60O C P 1 0 m P( 91 (0 P1m 1 ( P1 PC10 1C0m/1C 0m Sb3yTAcb7A45 ?7 3Rt For Ao ?ATFtbTA?F tlA Mo7 bA 7b 3y T 3Rtx3zG8a1 h2D1Cm0O 73ryoMF?byG8a1 9-ARFT)2GE h2DemeO3Rtx3z 73ryoMF?by9-ARFT)2GE i9n1eMb9 h2D10mCO3Rtx3z i9n1eMb9 73ryoMF?by pozAss h2DeP( O3Rtx3z 73ryoMF?bypozAss r 3 94 AGO2 binds to PREs and overlaps extensively with TrxG and PcG proteins Approximately 15% of AGO2 sites correspond to regions that can be regulated by both TrxG and Polycomb Group (PcG) proteins. The TrxG and PcG complexes maintain transcriptional activation or repression, respectively, of critical developmental regulators and are recruited by DNA-binding proteins that recognize Polycomb Response Elements (PREs), which are frequently juxtaposed to chromatin insulators (reviewed in Simon and Kingston 2009). This close configuration is particularly evident in the BX-C locus, in which insulators act as barriers to constrain PRE activity directionally. The high resolution afforded by ChIP-seq allows AGO2 detection specifically at all known PREs in the BX-C (bx, bxd, iab-2, Fab-6, Fab-7 , and Fab-8) despite their close proximity to insulators in this locus (Figure 3-1, 3-2A). Additionally, AGO2 associates with 84% of PREs across the genome, as previously defined (Oktaba et al. 2008; Sc hwartz et al. 2010) (Figure 3-2A, left panel). It should be noted that the probability-based enrichment values calculated for the AGO2 overlap with PREs and associated factors are higher than that with insulator proteins; this result is influenced by the small number of sites bound by PcG proteins genome-wide compared to insulator proteins (Figure 3-2B). Finally, mild but statistically significant overlap is also detected between AGO2 and annotated cis- regulatory modules (CRMs) in the REDFly database (Gallo et al. 2011), which is biased towards extensively studied TrxG and PcG regulated genes. AGO2 chromatin localization at PREs resembles that of TrxG proteins more closely than that of PcG proteins. Genome-wide, AGO2 overlaps extensively with the TrxG proteins Trx-N, Trx-C, and Ash1 as well as with the recruiter proteins Pho, Phol, 95 Sfmbt, Dsp1, and GAF, which also associate with non-PRE sites in the genome (Figure 3-2 (A-B)). Furthermore, AGO2 colocalizes substantially with the sharply peaking PRE- associated PcG proteins E(z), Ph, and Psc, as well as the broadly spreading Pc and H3K27me3; however, AGO2 itself does not bind chromatin in extended domains (Figure 3-1, 3-2A-B). Furthermore, AGO2 binds both Fab-7 and Fab-8 PREs in S2 cells, in which Abd-B is silent, as well as in S3 cells, in which Abd-B is expressed (Figure 3-1). Likewise, recruiter and TrxG proteins bind at Abd-B and its PREs irrespective of transcriptional expression state (Beisel et al. 2007), whereas PcG recruitment at Abd-B is only apparent in S2 cells (Breiling et al. 2004). This observation suggests that AGO2 does not require PcG proteins in order to associate with PREs. In order to obtain further insight into the specificity of AGO2 chromatin association, de novo motif analysis of AGO2 binding sites was performed. The analysis of the central 500 bp of 500 random AGO2 bi nding sites using the MEME algorithm (Bailey and Elkan 1995) identified a GA-rich consensus binding sequence reminiscent of the binding motif for the TrxG and insulator-associated GAGA-factor (GAF) (Figure 3- 4A) (Farkas et al. 1994; Belozerov et al. 2003; Schweinsberg et al. 2004). Similar results were obtained using all or non-GAF occupied AGO2 binding sites with the GADEM (Li 2009) and Weeder (Pavesi and Pesole 2006) algorithms (data not shown). 96 Figure 3-4. AGO2 behaves as a TrxG protein. (A) Highest-scoring de novo AGO2 binding site motif found by MEME using the center 500 bp of a random subset of 500 AGO2 binding site sequences. (B) Percentage of adult male flies displaying second and/or third legs with at least one ectopic sex comb tooth as an indication of posterior to anterior transformation was scored in the indicated genotypes, and number of flies (n) scored is shown. (C) Western blotting of AGO2, Pc, and Mod(mdg4)2.2 in wildtype and AGO251B adult male extracts. De novo AGO2 motif analysis was performed by Ryan K. Dale. 97 AGO2 Mod(mdg4)2.2 Pc AGO2 51 B WT B A Genotype n % transformation Pc4 / + 302 61.6 AGO2414 / + 357 0 AGO251B / + 337 0 +, Pc4 / AGO2414, + 376 44.1 +, Pc4 / AGO251B, + 449 36.6 +, Pc4 / AGO2V966M, + 278 61.9 AGO2V966M, Pc4 / AGO2V966M, + 339 62.0 C 98 AGO2 opposes Polycomb function Given the high overlap of AGO2 with TrxG proteins, we tested whether AGO2 affects either TrxG or PcG function. I anticipated that AGO2 may function as a trxG gene since the genes that encode GAF and Mod(mdg4)2.2 chromatin insulator proteins have been shown to behave as trxG genes (Farkas et al. 1994; Gerasimova and Corces 1998). I examined the classic posterior to anterior transformation phenotype of Pc4 /+ mutants and determined that 62% of adu lt males exhibit ectopic sex combs on second and/or third legs (Figure 3-4B). The AGO2414 /+ mutation results in a mild suppression of the Pc4 /+ phenotype such that a reduced number of double mutant males, 44%, display transformation. Interestingly, the partial loss-of-function AGO2414 /+ mutation is not defective for RNAi-dependent silencing in the heterozygous state (Okamura et al. 2004). Furthermore, heterozygous null AGO251B/+ mutants display stronger suppression of the Pc4 /+ phenotype in that only 37% of flies exhibit transformation. Neither AGO2414 /+ nor AGO251B/+ mutants, which both harbor dele tions of the first two exons of AGO2 (Okamura et al. 2004; Xu et al. 2004), exhibit developmental delays compared to wild type (data not shown). The AGO2V966M point mutation results in production of wildtype levels of catalytically inactive protein incompetent for RNAi-dependent silencing (Kim et al. 2007) but capable of associating with polytene chromosomes (data not shown). Importantly, the heterozygous AGO2V966M /+ or homozygous AGO2V966M mutations do not affect the Pc4 /+ phenotype, indicating that Slicer catalytic activity of AGO2 is not required for the suppression of the Pc4 /+ phenotype. This suppression is not due to an indirect effect on Pc gene expression as Pc protein levels are equivalent in wild type and 99 AGO251B mutants (Figure 3-4C). These results indicate that AGO2 behaves as a trxG gene and can counteract PcG function. In order to determine whether PcG affects AGO2 recruitment on chromatin, I performed ChIP analysis of the Abd-B locus in S2 cells depleted of Pc. Transfection of dsRNA corresponding to Pc results in reduction of the target protein by over 90% (Figure 3-5A). I first analyzed Trithorax (TRX) and H3K27ac association with chromatin in mock-treated cells. In S2 cells, where Abd-B is PcG-repressed, TRX is present at S2 cells at baseline levels equivalent to Rpl32 (Figure 3-5B). H3K27ac histone mark, which is associated with TrxG activity, is detected at IgG negative control levels. These observations are consistent with published data (Schwartz et al. 2010). AGO2 ChIP results in an approximate 12-fold enrichment of Fab-7 (set 2), eight-fold enrichment of Fab-8 PRE (set 7), four-fold enrichment of the Abd-B RE promoter (set 14) and six-fold enrichment of an intronic site (set 11) compared to RpL32, which shows low AGO2 association. In Pc-depleted cells, TRX and H3K27ac association with chromatin is increased. AGO2 recruitment to most sites is increased approximately 1.5 to three-fold across the entire Abd-B locus. Therefore, similarly to TrxG proteins, increased AGO2 association with chromatin is correlated with derepression of target genes caused by the reduction in Pc levels. I also examined whether TrxG proteins affect AGO2 association with chromatin by performing AGO ChIP of the Abd-B locus in S3 cells depleted of TRX. TRX protein levels were reduced using dsRNA RNAi by over 90% (Figure 3-5C). In mock-treated S3 cells, where Abd-B is transcribed, ChIP of Pc and H3K27ac revealed baseline levels of Pc and strong H3K27ac signal over th e entire locus (Figure 3-5D ). AGO2 association with 100 chromatin is similar to S2 profile. TRX KD correlated with increased Pc association and loss H3K27ac over the Abd-B locus. Interestingly, no change in AGO2 association with chromatin was detected upon TRX depletion. Therefore, TrxG proteins do not affect AGO2 recruitment to chromatin. Taken together with AGO2 ChIP in Pc-depleted S2 cells, these results suggest that AGO2 may associate with open chromatin possibly to promote transcription. 101 Figure 3-5. Effects of Pc and TRX knockdown on AGO2 association with chromatin. (A) Western blotting of lysates from S2 cells mock treated (lane 1) or transfected with Pc dsRNA (lane 2). (B) S2 cells mock treated (light blue) or transfected with Pc dsRNA (dark blue) were subjected to ChIP using ?-TRX-N, ?-H3K27ac and ?-AGO2 antibodies. Locations of primer sets are indicated in Figure 3-1. Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. IgG negative control IPs for all sites yielded <0.06% input. (C) Western blotting of lysates from S3 cells mock treated (lanes 1) or transfected with TRX dsRNA (lane 2). (D) S3 cells mock treated (light green) or transfected with Pc dsRNA (dark green) were subjected to ChIP using ?-Pc, ?-H3K27ac and ?-AGO2 antibodies. Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. IgG negative control IPs for all sites yielded <0.05% input. 102 ?-TRX ?-H3K27Ac ?-H3K27Ac ?-Pc ?-AGO2 ?-AGO2 0 5 10 15 20 25 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input S 3 Mo c k S 3 TRX K D 0 0. 5 1 1. 5 2 2. 5 3 3. 5 4 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input 0 5 10 15 20 25 30 35 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input 0 1 2 3 4 5 6 7 8 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input S 2 Mo c k S 2 Pc K D B D Mock S2Pc KD CP190 AGO2 Pc A TRX AGO2 Lamin Mock S3TRX KD C 1 2 1 2 0 0. 5 1 1. 5 2 2. 5 3 3. 5 4 4. 5 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input 0 0. 5 1 1. 5 2 2. 5 3 3. 5 Rp l 32 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 % Input 103 AGO2 but not its catalytic activity is specifically required for Fab-8 insulator activity Given the high overlap of AGO2 with insulator sites throughout the genome, particularly of the CP190 class, I wished to determine whether AGO2 is required for activity of the well-characterized CTCF/CP190 dependent insulator Fab-8 of the Abd-B locus. I utilized a transgenic enhancer blocking assay in which a genomic fragment containing the Fab-8 insulator and PRE positioned between a mini-white (mini-w+ ) reporter and w+ enhancer reduces reporter expression, resulting in intermediate levels of pigmentation in the adult eye (Barges et al. 2000). Compared to wild type, AGO2414 /+, AGO251B/+, AGO2 414 , and AGO251B mutants carrying the Fab-8 insulator transgene display increased eye pigmentation corresponding to the strength of AGO2 loss-of- function mutation, indicating a positive role for AGO2 in Fab-8 insulator function (Figure 3-6A). Importantly, the AGO2V966M catalytic activity mutant remains fully competent for Fab-8 insulator activity. In comparison, loss-of-function CP1904- 1/CP190H31-2 mutants (Pai et al. 2004) that reduce Fab-8 insulator function (Gerasimova et al. 2007) display a more modest increase of mini-w+ expression than AGO251B/+ mutants (data not shown). No differences compared to wild type are detected in AGO2 mutant flies carrying a transgene containing only the Fab-8 PRE or no cis-regulatory sequence (Figure 3-6A), indica ting that the effects on the Fab-8 insulator reporter are likely specific to the insulator. Importantly, comprehensive genetic analysis of RNA silencing mutants revealed that AGO2, but not other RNA silencing factors, is required for Fab-8 insulator activity (data not shown, Moshkovich et al. 2011). 104 In order to obtain mechanistic insight into the possible function of AGO2 with respect to Fab-8 insulator activity, the in vivo localization of insulator proteins in AGO2 mutants was examined. Previously it was shown that positive or negative effects of certain RNA silencing mutants on gypsy insulator activity correlate with the integrity of insulator bodies (Lei and Corces 2006). The AGO251B null mutation does not appear to reduce Fab-8 function by disrupting the integrity of insulator bodies (Figure 3-6B). Furthermore, no overall differences in the ability of CTCF and CP190 to associate with chromatin or specifically with the BX-C on polytene chromosomes of wild type compared to AGO251B mutants were observed (Figure 3-6C ). Finally, Western blotting of wild type and AGO251B mutants indicates no effect on CTCF or CP190 protein levels (Figure 3-6D). 105 Figure 3-6. AGO2 but not its catalytic activity is required for Fab-8 insulator function. (A) Eye color due to expression of a transgenic construct carrying no regulatory element (top row), Fab-8 insulator and PRE (middle row) or Fab-8 PRE (bottom row) between the mini-white enhancer and its coding sequence in wildtype, AGO2414 /+, AGO251B/+ AGO2414 , AGO251B, and AGO2V966M flies. (B) Visualization of insulator bodies by indirect immunofluorescence of whole mount larval imaginal discs using ?-CP190 antibodies (red) merged with DAPI staining (blue) in wild type and AGO251B mutants. (C) Polytene chromosome staining of ?-CTCF (green), ?-CP190 (red), and merged images in wildtype and AGO251B mutants. Arrows point to the BX-C locus. (D) Western blotting of CP190, CTCF, and Pep (loading control) in wildtype and AGO251B pupal extracts. 106 Empty vector Fab-8 Insulator + PRE Fab-8 PRE WT AGO251B/+ AGO251BAGO2414/+ AGO2414 AGO2V966MA C AGO 251 B CP190 CTCF Pep B CP190CTCF Merge AGO251B D AGO251B CP190 WT W T WT 107 AGO2 interacts physically with CTCF and CP190 In order to address whether AGO2 influences chromatin insulator activity in a direct manner, its subcellular localization compared to that of CP190 was examined. In S2 cells, CP190 localization is mainly diffuse within the nucleus whereas in S3 cells, CP190 is nuclear but also concentrates into insulator bodies reminiscent of those seen in larval imaginal disc cells (data not shown; Moshkovich et al. 2011). In both S2 and S3 cells, AGO2 localizes throughout the cell but concentrates preferentially in the nucleoplasm in the majority of cells. Nuclear signal is reduced upon siRNA knockdown of AGO2 (data not shown). Importantly, AGO2 staining is excluded from the heterochromatic DAPI dot and is mainly nonoverlapping with the heterochromatin protein HP1 (data not shown). Next, physical interactions between insulator complexes and RNA silencing components were probed for. Immunoprecipitation of AGO2 from embryonic nuclear extracts at high monovalent salt concentrations results in copurification of CTCF and CP190 but not the gypsy insulator protein Mod(mdg4)2.2 (Figure 3-7A). In addition, column-based immunoaffinity purification of CP190-associated complexes from nuclear extracts verifies the presence of core gypsy and Fab-8 insulator components CP190, Su(Hw), Mod(mdg4)2.2, and CTCF, and reveals association of the RNA silencing components Rm62, Piwi, and AGO2 (Figure 3-7B ). Interactions between insulator proteins, Piwi, or AGO2 with CP190 complexes are not affected by RNaseA treatment under conditions that disassociate Rm62 (Fi gure 3-7C), suggesting that RNA does not mediate physical associations between Piwi or AGO2 and CP190. Physical interactions 108 between these RNA silencing components and CP190, either direct or in the context of larger complexes, are consistent with the direct involvement of Piwi and Rm62 in gypsy insulator activity and that of AGO2 in CTCF/CP190 insulator activity. 109 Figure 3-7. AGO2 associates physically with CP190 and CTCF. (A) Western blotting of embryonic nuclear extracts immunoprecipitated with ?-AGO2 antibodies. Nuclear extract (lane 1) bound to control IgG (lane 2) or ?-AGO2 immobilized on ProtA-sepharose (lane 3) at > 1.1 M monovalent salt concentration. (B) Western blotting of embryonic nuclear extracts (lane 1) bound to a control preimmune column (lanes 2-4) or ?-CP190 column (lanes 5-7) and step eluted with increasing MgCl2 concentrations as indicated. (C) Western blotting of embryonic nuclear extracts (lane 1) bound to ?-CP190 columns either untreated (lanes 2-4) or treated (lanes 5-7) with RNaseA and step eluted with increasing MgCl2 concentrations as indicated. Immunoaffinity purification with ?-CP190, RNaseA treatment, and immunoprecipitation with ?-AGO2 was performed by Elissa P. Lei. 110 Pre-immune ??CP190 0.01 0.25 1 0.01 0.25 1 M MgCl 2 CP190 CTCF Piwi Rm62 AGO2 Su(Hw) Mod(mdg4)2.2 Nuclear extrac t 1 2 3 4 5 6 7 ?? AGO 2 IgG CP190 AGO2 1 2 3 CP190 Piwi Rm62 AGO2 Su(Hw) - RNaseA 0.01 0.25 1 0.01 0.25 1 M MgCl 2Nuclear extrac t + RNaseA B C 1 2 3 4 5 6 7 Nuclear extrac t Mod(mdg4)2.2 CTCF A 111 AGO2 associates with chromatin downstream of CTCF and CP190 In order to examine whether AGO2 recruitment to chromatin is downstream to that of CTCF and CP190, chromatin association of these insulator proteins in the absence of AGO2 was examined. No changes in CP190 or CTCF recruitment in AGO2 knockdowns were observed; however, a significant amount of residual AGO2 remains on chromatin despite at least 90% depletion of total AGO2 (data not shown). As a more rigorous test, I examined AGO251B null mutants derived from mothers with AGO251B ovaries by deriving germline clones; these mutants contain no maternal or zygotic protein. ChIP was performed on adult heads of AGO251B/+ or AGO251B mutant siblings derived from the germline clones as well as from wild type flies. ChIP profiles of CTCF and CP190 in adult head tissue are similar to that observed in S2 and S3 cells but with considerable enrichment at the Fab-7 insulator (Figure 3-8A, pr imer set 2). Importantly, no changes were observed in AGO251B null mutants compared to heterozygous siblings or to wild type. Pc chromatin association is also unchanged in AGO251B null mutants (data not shown). These results in combination with the finding that CP190 and CTCF localization is unchanged in polytene chromosomes of AGO251B null mutants (Figure 3- 6B), suggest that AGO2 is not required for CTCF or CP190 recruitment. 112 Figure 3-8. AGO2 is requi red for looping at the Abd-B locus. (A) CTCF and CP190 chromatin association is unaffected in AGO251B null mutants. Adult heads of wild type (blue) as well as AGO251B/+ (red) or AGO251B (green) derived from AGO251B germline clones were subjected to ChIP using ?-CP190, ?-CTCF, and ?- Pc antibodies. Locations of primer sets are indicated in Figure 3-11B. Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. (B) 3C looping interactions between cis-regulatory elements of the Abd-B locus are dependent on AGO2. Relative interaction frequencies between EcoRI restriction fragments (triangles) and anchor regions (red vertical lines) are shown for wild type (open circles), CP190P11/CP190H31-2 (filled red circles), CTCFy+2 (filled green circles) and AGO251B (filled orange circles) mutant larval brains and imaginal discs. 113 ? ? ? ? ? ? ??? ??? ??? ??? ??? ??? ??? ??? ??? ?? ??? ?? ??? ??? ??? ?? ??? ??? ?? ??? ??? ?? ??? ??? ?? ??? ??? ?? ???? ??? ?? ?? ??? ?? ??? ??? ? ?? ??? ??? ? ???? ??? ?? ?? ??? ?? ???? ????? ???? ???? ? ?? ???????? ???? ???? ??? ?? ?? ?? ?????????????? ???????? ???????? ???????? ???????? ???????? ???????? ???????? ???????? ???????? ???????? ????? ????????? ?????????? ????????? ?? ??? ???? ??? ????? ? ?? ??? ??? ??? ? ??? ??? ???????????? ??? A B 0 3 6 9 RpL32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 % input WT AGO251B/+ AGO251B?-CP190 0 2 4 6 8 RpL32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 % input ?-CTCF 114 CP190, CTCF and AGO2 are required for looping at the Abd-B locus AGO2 association with non-insulator regions of Abd-B in CP190 and CTCF knockdowns is lost and may be the result of changes in looping interactions at this locus (data not shown, Moshkovich et al. 2011). In order to determine whether CP190 or CTCF insulator proteins mediate these or other long-range interactions in this locus, I examined locus-wide interactions by 3C in diploid larval brains and imaginal discs of wild type compared to CTCF (Gerasimova et al. 2007) and CP190 null mutants. These tissues represent a mixed population with a minority of cells expressing Abd-B. We scanned pair wise interactions using available EcoRI restriction sites in an 80 kb region encompassing the Fab-7 insulator to the most distal Abd-B RE promoter. Using an anchor at the Abd-B RB promoter, high levels of interaction are observed between the Abd-B RB promoter anchor and Fab-7 , Fab-8, and iab-8 enhancer in wild type (Figure 3- 8B). These interactions are de creased 1.5 to two-fold in both CP190P11/CP190H31-2 mutants and further decreased in CTCFy+2 null mutants. In addition, using an anchor at Fab-8, interactions with Fab-7 and the Abd-B RB promoter are decreased approximately two-fold in CP190 mutants with a greater decrease in CTCF mutants compared to wild type. These results indicate a requirement for both CP190 and CTCF for looping interactions between insulators, PREs, enhancers, and promoters of the Abd-B locus. I also addressed the possibility that AGO2 is required for insulator-dependent looping interactions at Abd-B. Similar to CP190 and CTCF mutants, 3C in diploid larval brains and imaginal discs of AGO2 null mutants from germline clones demonstrated high levels of interaction between the Abd-B RB promoter anchor and Fab-7 , Fab-8, and iab-8 115 enhancer in wild type (Figure 3-8B). These interactions are decreased 1.5 to two-fold in AGO251B mutants. High frequency interactions between the Fab-8 anchor and Fab-7 and the Abd-B RB promoter are decreased approximately two-fold in AGO2 compared to wild type, suggesting that AGO2 is required for CTCF/CP190 insulator-dependent looping interactions at Abd-B. The looping interactions, mediated by both AGO2 and the insulator proteins, may be required for proper expression of Abd-B (data not shown; Moshkovich et al. 2011). 116 DISCUSSION Here, I provide the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is downstream of CTCF and CP190 binding and may be partially achieved via looping interactions among cis- regulatory regions and promoters. I propose that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Figure 3-9). AGO2 localizes predominantly to euchromatin and not heterochromatin The presented results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 in polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in non-repetitive euchromatic but not repeat-rich regions as determined by genome-wide ChIP-seq. I suggest that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the posttranscriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function. 117 Figure 3-9. Model for AGO2 function with respect to CTCF/CP190 chromatin insulator activity. Looping at the Abd-B locus between the Fab-8 insulator and Abd-B promoter is dependent on CTCF/CP190 insulator interactions. This specialized configuration promotes interactions between Fab-8 associated cis-regulatory elements and the promoters to facilitate proper gene expression. AGO2 is recruited downstream of CTCF/CP190 chromatin association and acts to either promote or stabilize looping interactions. Transfer of AGO2 to non-insulator sites may be achieved through CTCF/CP190-dependent looping interactions. 118 RNAi-independent function for AGO2 at chromatin Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2 (Chung et al. 2008; Czech et al. 2008 ; Ghildiyal et al. 2008; Kawamura et al. 2008; Okamura et al. 2008). Second, AGO2 but not Dcr-2 is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity. An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. It was determined that CP190 mutants remain competent for silencing using a GMR-wIR hairpin transgene (Lee et al. 2004), suggesting that AGO2 chromatin association is not required for RNAi (data not shown). Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated. Role of AGO2 in Fab-8 insulator function 119 AGO2 but not other RNA silencing factors exerts a unique positive role in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wildtype levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis. I show for the first time that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, we find that the Abd-B RB promoter interacts frequently with Fab-7 , Fab-8, and the iab-8 enhancer and moreover that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus. AGO2 is recruited to chromatin insulator sites as well as non-insulator sites in a CTCF/CP190-dependent manner. I speculate that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping 120 mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions (Comet et al. 2011). Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190- dependent looping interactions. Role of AGO2 in long range chromosomal interactions AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities (Gerasimova et al. 2007). However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis- regulatory elements that can maintain looping interactions of this locus (Bantignies et al. 2011), suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body. AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2 (Grimaud et al. 2006). I found that the AGO251B null mutation has no effect on Fab-X PRE pairing-dependent silencing 121 on sd as assayed in that study (data not shown), and the genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements, that long- range interactions are dependent on insulator sequences and not PREs (Li et al. 2011). Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures. Conclusions It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In C. elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA (Guang et al. 2010). Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation (Claycomb et al. 2009; van Wolfswinkel et al. 2009). Recently, it has been shown that S. pombe Ago1 participates in surveillance mechanisms to prevent read-through transcription of mRNA (Gullerova and Proudfoot 2008; Zofall et al. 2009; Halic and Moazed 2010). However, the majority of Ago1 associates with 122 heterochromatic regions (Noma et al. 2004), and it is not clear thus far if Ago1 directly associates with euchromatin or acts posttranscriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms. 123 ACKNOWLEDGEMENTS We thank A. Beyer for ?-Pep, F. Fuller-Pace for ?-p68, J. Kassis for ? -Pho, Q. Liu, A. Mueller, and M. Siomi for ?-AGO2, P. O?Farrell and D. Moazed for ?-Pc; and C. Berg, J. Birchler, R. Carthew, V. Corces, G. Hannon, S. Hou, F. Karch, J. Kassis, G. Shanower, P. Schedl, and P. Zamore for strains. We are indebted to P. Murphy for antibody characterization and M. Emmett for primers; A. Dean for 3C protocols; S. Grewal, F. Karch, and B. Oliver for discussions; and J. Kassis and L. Matzat for critical reading of the manuscript. 124 AUTHOR CONTRIBUTIONS Elissa P. Lei Performed immunoaffinity purification with ?-CP190 and RNaseA treatment. Performed immunoprecipitation with ?-AGO2. Performed immunostaining of salivary gland polytene chromosomes and S2 and S3 cell staining. Contributed to the writing of the manuscript presented in this chapter. Ryan K. Dale Performed AGO2 ChIP-seq analysis, AGO2 binding site comparisons with other datasets, de novo AGO2 motif analysis and siRNA analyses. Generated Figures 3-1, 3-2, 3-3 and 3-4A. Parul Nisha Performed whole mount immunofluorescence staining of insulator bodies. Patrick J. Boyle Designed primers for ChIP. Brandi A. Thompson Designed primers for 3C and prepared 3C control template. 125 MATERIALS AND METHODS Drosophila strains Flies were maintained on standard cornmeal medium at R.T. or 25 ?C. Newly eclosed flies were collected and aged for 24-27 h and examined for eye pigmentation. Larvae for immunostaining of imaginal discs were raised at 25 ?C. Larvae for immunostaining of polytene chromosomes were raised at 18? C. The Fab-8 Insulator + PRE transgene contains a HindIII-EcoRI fragment, and the Fab-8 PRE transgene contains an EcoRI-AflII fragment (Barges et al. 2000). Transgenes were scored as single copy. The AGO251B/+ mutation was tested on five independent Fab-8 Insulator + PRE insertion lines, and similar results were observed. Homozygous AGO251B flies exhibit a high degree of male and female sterility, but these phenotypes appear to be caused by second site mutations unlinked to the AGO2 mutation. Furthermore, AGO251B mutants exhibit a low, variable level of protein likely maternally deposited. Consequently, homozygous mutant germline clones were produced by recombining the AGO251B mutation with FRT2A and inducing recombination with a ovoD1 marked FRT2A chromosome using a hs-FLP recombinase induced for 1h in larvae at 5 d and 6 d of age as described pr eviously (Selva and Stronach 2007). These flies were then crossed to AGO251B/+ males to obtain the desired progeny. The progeny were verified by Western blotting, PCR, and ChIP, and the same results were obtained with AGO2321 /AGO2454 null mutants (Hain et al. 2010) from AGO2321 germline clones (data not shown). 126 Indirect immunofluorescence Preparation and immunostaining of salivary gland polytene chromosomes was performed as described previously (Lei and Corces 2006). Cell staining and whole mount staining are detailed in Supplemental Experimental Procedures. Rabbit ?-Su(Hw) (Moshkovich and Lei 2010) and guinea pig ?-CP190 (generated similarly as in (Pai et al. 2004)), rabbit ?-CP190 (Pai et al. 2004), rat ?-CTCF (Gerasimova et al. 2007), rabbit ? - CTCF (Gerasimova et al. 2007), mouse ?-AGO2 (9D6) (Kawamura et al. 2008), and rabbit ?-Pc antibodies (a kind gift from D. Moazed and P. O?Farrell, UCSF) were used for staining. Samples were mounted with Vectashield (Vector Labor atories), and images were acquired on a Leica DM5000 epifluorescence microscope with Openlab (Perkin Elmer) software. Western blotting Lysates from whole pupae, the anterior third of third instar larvae, whole flies, or cell lines were prepared as described previously (Lei and Corces 2006). Guinea pig ? - CP190, rabbit ?-CP190, (generated similarly as in (Pai et al. 2004)), guinea pig ?- Su(Hw) (generated similarly as in (Moshkovich and Lei 2010)), guinea pig ?- Mod(mdg4)2.2 (Moshkovich and Lei 2010), rabbit ?-CTCF, rat ?-CTCF, mouse ?-Pep (Amero et al. 1991), MAD1 mouse ?-p68 (Ishizuka et al. 2002), mouse ?-AGO2 (4D2) 127 (Okamura et al. 2004), mouse ?-AGO2 (9D6), rabbit ?-Piwi (Abcam ab-5207), rabbit ?- AGO2 (Abcam ab-5072), mouse ?-Lamin (ADL67.10) (Stuurman et al. 1996), rabbit ?- Pc and rabbit ?-TRX-C (Schuettengruber et al. 2009) were used for Western blotting. Specificity of generated and commercial antibodies was verified by blotting mutant fly lysates and/or cells knocked down with the corresponding dsRNA. Immunoaffinity purification Immunoaffinity purification with ?-CP190 and RNaseA treatment was carried out as described previously (Lei and Corces 2006). Immunoprecipitation with ?-AGO2 (9D6) was performed using nuclei isolated from 20 g of 0-24 h embryos as described previously (Lei and Corces 2006). Nuclei were lysed by sonication in 5 mL HBSMT- 0.3% + 1 M KCl (50 mM HEPES, 150 mM NaCl, 1 M KCl, 3 mM MgCl2, 0.3% Triton X-100 (vol/vol), pH 7) including 1 mM PMSF and Complete protease inhibitor cocktail (Roche) and extracts prepared as described previously (Lei and Corces 2006). 1.2 mL of extract was bound overnight at 4?C to 1 mL ?-AGO2 (9D6) tissue culture supernatant or 1.4 ?g mouse IgG (Santa Cruz) prebound to rProtA-sepharose for 1 h at 4?C. Beads were washed three times with HBSMT-0.3% + 1 M KCl then once with HBSM (50 mM HEPES, 150 mM NaCl, 5 mM KCl, 3 mM MgCl 2), and eluted with denaturing sample buffer by boiling for 5 min. Samples were West ern blotted as described previously (Pai et al. 2004). Chromatin immunoprecipitation and ChIP-seq 128 Preparation of ChIP samples and analysis was performed essentially as described previously (Moshkovich and Lei 2010). S2 and S3 cells were grown at 25 ?C in Shield and Sangs M3 Insect medium (Sigma) supplemented with 0.1% yeast extract, 0.25% bactopeptone, and 10% fetal bovine serum (HyClone). Immunoprecipitations were performed with ?-AGO2 (9D6), rabbit ?-CP190 (this study), and rabbit ?-CTCF, rabbit ?-Pho (Fritsch et al. 1999), rabbit ?-Pc, rabbit ?-TRX N-terminal (Schwartz et al. 2010), rabbit ?-H3K9ac (Abcam, ab4729), mouse IgG, and rabbit IgG (Santa Cruz Biotechnology) coupled to rProtein A agarose beads (GE Healthcare). Similar results but with lower signal were obtained with rabbit ?-AGO2 (Jiang et al. 2005), rabbit ?-AGO2 (Meyer et al. 2006) or with ?-Flag (M2, Sigma) or ?-HA (12CA5, Santa Cruz) using chromatin prepared from HA/Flag-AGO2 transgenic flies also expressing wildtype AGO2 (Czech et al. 2008). Primers used are indicated in Supplemental Table S3. Samples for ChIP-seq from input DNA and AGO2 ChIP were prepared according to the manufacturer?s protocol (Illumina). DNA was sequenced on an Illumina Genome Analyzer at the NIDDK Genomics Core. Computational methods are detailed in Supplemental Materials and Methods. AGO2 ChIP-seq data are available at GEO (GSE22623). AGO2 ChIP-seq analysis 36 bp sequenced tags were mapped to the D. melanogaster genome (dm3) with Bowtie (Langmead et al. 2009), retaining uniquely mapping reads with up to 2 129 mismatches in the first 28 bp (-m1 -n2 --best) . Binding sites were identified using MACS 1.3.7.1 (Zhang et al. 2008) (--tsize 36 --bw 150). The resulting peaks were filtered to retain only those peaks with a false discovery rate of 5% or below. To assess the possible enrichment of AGO2 binding in heterochromatin, we separated the reads into uniquely mapping (i.e., euchromatic) and multiply mapping (i.e., repetitive sequence or heterochromatin) and asked whether the ratio of unique to multiply mapping reads was different in IP versus input using a chi-square test. AGO2 binding site comparisons with other datasets ChIP-chip tiling array data were downloaded from GEO, ArrayExpress, or supplemental material as available (Schwartz et al. 2006; Czech et al. 2008; Kawamura et al. 2008; Lee et al. 2008; Oktaba et al. 2008; Bushey et al. 2009; Schuettengruber et al. 2009; Negre et al. 2010; Schwartz et al. 2010) as described in Table S2 and stored as a collection of BED-format files. Where possible, called peaks from the original study were used to retain consistency with published work. Otherwise the peak-calling algorithm described by the authors was implemented to obtain binding sites. BED files were mapped from dm2 to dm3 if necessary with the liftOver tool from UCSC (http://genome.ucsc.edu/cgi-bin/hgLiftOver) and were filtered to remove features from the heterochromatic chromosomes since some data sets only considered the euchromatic chromosomes. Promoters were defined as 250 bp upstream from the transcription start site (TSS) of transcripts (mRNA, tRNA, rRNA, snoRNA, snRNA, miRNA) annotated in FlyBase r5.29. The "AGO2 minus GAF" BED file was created by removing all sites in 130 the AGO2 S2 BED file that overlapped by at least 1 bp with the GAF S2 sites. The TrxG (all Ash1, Trx-C, and Trx-N sites), PcG (all E(z), H3K27me3, Pc, Ph, PRE, and Psc sites), and insulators (all BEAF-32, CTCF, CP190, and Su(Hw) sites) BED files were created by concatenating all sites together and merging into non-redundant sites. 7747 active promoters were defined in S2 cells as having both H3K4me3 and PolII 250 bp upstream or 750 bp downstream from the TSS. 7281 inactive promoters were defined as having neither factor in this same window. H3K4me3 data from S2 cells were obtained from (Negre et al. 2010) (GEO accession GSM409457). PolII data from S2 cells were obtained from modENCODE consortium (modMine.org). Unique transcription start sites for all annotated RNAs were retrieved from FlyBase r5.29 and provided to CEAS (Shin et al. 2009) to calculate a profile for IP and input separately. The resulting profiles were scaled by library size, and the input profile was then subtracted from the IP profile to obtain the final scaled, input-subtracted profile. Profiles for transcription termination, active promoters, and inactive promoters were calculated similarly. For the binary heatmap, supervised hierarchical clustering of overlap by at least 1 bp was performed as in (Kim et al. 2008). For the colocalization heatmap, permutation tests were performed to assess the degree of overlap between binding sites of each pair of factors similar to a previous study (Negre et al. 2010). Specifically, for each pair of factors A and B, the number of features in A that overlapped with at least one feature in B was calculated using BEDtools v2.6.1 (Quinlan and Hall 2010). Then features in A were randomly shuffled within each chromosome, and intersections with B were again calculated. This was repeated 1000 times for each pair of factors, resulting in a null 131 distribution of intersections. An empirical p-value was calculated as the percentile of the original intersection count within this distribution, and the enrichment score is defined as original intersection count divided by the median of the null distribution. The matrix of pairwise enrichment scores was clustered using complete linkage using correlation as the distance metric (as implemented in scipy.cluster in the SciPy package for Python). De novo motif analysis MEME (Bailey and Elkan 1994) v4.5.0 was used for de novo motif finding. The center 500 bp were extracted from a randomly selected set of 500 of the 2084 S2 AGO2 peaks. DNA mode was used with a maximum number of 3 iterations, a maximum width of 15 bp, up to 3 motifs, allowing zero or one motif per sequence, and in reverse- complement mode (parameters -dna -maxiter 3 -maxw 15 -nmotifs 3 -revcomp -mod zoops). Similar motifs were found with GADEM using all 2084 s ites and the center- weighted option and Weeder using the center 500 bp of 500 random AGO2 sites and default parameters. siRNA analyses Data were obtained from GSM266 765 (Kawamura et al. 2008), GSM280087 (Czech et al. 2008), GSM272652 (Okamura et al. 2008) and GSM239051 (Ghildiyal et al. 2008) and pooled. NLS-P19 data (Fagegaltie r et al. 2009) were processed separately. Adapters were removed from reads before being mapped to the dm3 assembly with 132 Bowtie, allowing only uniquely-mapping reads and allowing up to one mismatch in the first 28 bp (parameters -m 1 -k 1 -n 1 --best --strata). Reads were then size-filtered, retaining only those 19-22 nt in length, and further filtered by removing those overlapping by 1 or more bp with annotated mirBase miRNA sites (mirbase.org) (Griffiths-Jones et al. 2008) . Reads falling in the white gene were also removed. Finally, PCR bias was removed with Picard's Ma rkDuplicates program (Li et al. 2009b), collapsing duplicate reads into a single read. For the heatmaps, siRNA reads were then clustered according to a previously described algorithm (Czech et al. 2008). For the cumulative histograms, all siRNA reads retained after the PCR-filtering step were used, and their overlap was computed with all possible 3? cis-NATs, as previously reported (Okamura et al. 2008) , and divided by the length of each 3? cis-NAT to obtain an siRNA density for each feature. Random backgrounds were calculated by averaging the histograms of 1000 randomizations, where each randomization consisted of intrachromosomal shuffling of sites bound by a particular factor while keeping the siRNA reads fixed. siRNA densities were similarly calculated for PolII, H3K27me3, and AGO2. Double stranded RNA and siRNA knockdowns Amplicons used for dsRNA knockdowns were designed based on recommendations from the Drosophila RNAi Screening Center. Templates were PCR amplified from genomic DNA using primers containing the T7 promoter sequence. dsRNAs were produced by in vitro transcription of PCR templates using the MEGAscript T7 kit (Ambion) and purified using NucAwa y Spin Columns (Ambion). Transfections 133 using 200 ng-1.25 ? g of dsRNA or 100 pmol of siRNA per million cells, or no dsRNA/siRNA for mock treatment were performed using Cellfectin (Invitrogen), Effectene (Qiagen), or Cell Line Nucleofect or Kit V (Amaxa Biosystems) transfection reagent using the recommended protocol. Four to six days after transfection, cells were collected, and knockdown efficiency was confirmed by Western blotting. Highest knockdown efficiencies were generally obtained using the Amaxa system. No differences were seen with mock treatment, GFP dsRNA, or luciferase dsRNAs. Primers used are indicated in Supplemental Table S3, and 3C methods are detailed in Supplemental Materials and Methods. Cell staining S2 and S3 cells were prepared for staining by washing in PBS and affixed to poly- L-lysine coated slides for 10 min at R.T. Cells were fixed in 4% paraformaldehyde + 0.1% Triton X-100 in PBS for 30 min and then blocked in 10% normal goat serum + 0.1% Triton X-100 in PBS for at least 30 mi n. Cells were incubated with rabbit ?-CP190 (Pai et al. 2004) and mouse ?-AGO2 (9D6) including 0.1% Triton X-100 overnight at 4?C, washed 3 times in PBS + 0.1% Triton X-100 (PBS-X-0.1%) for 15 min at R.T., and incubated with Alexa594 anti-rabbit and Al exa488 anti-mouse antibodies (Invitrogen) in PBS-X-0.1% + 10% normal goat serum for 2 h at 37 ?C. Samples were washed 3 times in PBS-X for 15 min at R.T., and stained with 100 ng/mL DAPI in PBS at R.T. for 1 min. Whole mount staining 134 For whole mount immunofluorescence staining of insulator bodies, brain and imaginal disc complexes from third instar larvae were dissected in PBS and fixed in 4% paraformaldehyde in PBS + 0.1% Tween (PBS-Tw) for 20 min on a rotating wheel. Samples were rinsed three times in PBS-Tw and blocked for 2 h in PBS + 0.3% Triton X- 100 (PBS-X-0.3%) + 10% normal goat se rum then incubated with rabbit ?-CP190 (Pai et al. 2004) with blocking solution overnight at 4?C on a rotating wheel. Samples were washed 3 times for 5 min each with PBS-X-0.3% followed by 3 times for 20 min in the same buffer on a rotating wheel at R.T. Alexa594 anti-rabbit antibody in PBS-X-0.3% + 10% normal goat serum was added for 2 h at 37 ?C on a rotating wheel. Samples were washed 3 times for 5 min with PBS-X followed by 3 times for 20 min, followed by one PBS-Tw wash for 5 min. Samples were stained with 100 ng/mL DAPI in PBS at R.T. for 5 min, followed by a PBS-Tw wash for 5 min. Chromosome Conformation Capture (3C) Drosophila S2 cells used for the 3C assays were grown in M3+BYPE (10% FBS). Before crosslinking, 4?10 6 cells were resuspended in 5 mL of fresh media. Crosslinking was performed by adding formaldehyde directly to the media at final concentration of 1% and incubating for 10 min at R.T. Reactions were quenched by adding glycine to a final concentration of 0.125 M and incubating for 5 min at RT. Reactions were incubated on ice 5 min followed by centrifugation at 1200 rp m at 4?C for 5 min. Cells were then washed with 5 mL of cold PBS and centrif uged at 1200 rpm at 4?C for 5 min. Lysis was 135 performed by incubating cells in 1 mL of Lysis buffer [10 mM NaCl, 0.2% NP-40, 10 mM Tris pH=8, protease inhibitors [1 Mini Complete tablet (Roche) per 10 mL Lysis buffer)] at 37?C for 20 min. Samples were then centrifuged at 4200 rpm at 4?C for 5 min, and the lysis step was repeated once. Brains and imaginal discs were dissected from ten male and ten female third instar larvae in Schneider?s S2 medium without serum and immediately centrifuged for 30 s at 6000 rpm. Pellets were resuspended in 700 ?l of Fixing buffer (50 mM HEPES pH 7.6, 100 mM NaCl, 0.1 mM EDTA , 0.5 mM EGTA) and 100 ?l 16% paraformaldehyde and rocked for 15 min at R. T. Reactions were quenched by adding 1 mL Stop solution (PBS, 0.01% TritonX-100, 0.125 M glycine) and rocked for 10 min at R.T. Reactions were washed with 1 mL Wash solution (50 mM Tris, 10 mM EDTA, 0.5 mM EGTA, 0.25% TritonX-100) twice rocking for 10 min. Pellets were homogenized with 200 ?l of Lysis buffer using a motorized pellet pestle. 800 ?l of Lysis buffer was added, and samples were incubated at 37? C fo r 20 min. Samples were then centrifuged at 4200 rpm at 4?C for 5 min, and resuspended in 1 mL Lysis buffer and incubated another 20 min at 37?C. Pellets from cell or larval samples were then washed with 0.8 mL of digestion buffer [0.2% NP-40, 1X NEBuffer 3 (NEB)] and centrifuged at 4200 rpm at 4?C for 5 min. Nuclei were resuspended in 1.6 mL of digestion buffe r with SDS added to a final concentration of 0.1% and incubated at 65? C for 30 min. Triton X-100 was added to a final concentration of 1% and incubated at 37?C for 15 min. A 40 ?L aliquot of the sample was taken and used as the undigested control. The remaining sample was digested with 1600 U of EcoRI (NEB) at 37?C O/N. Samples were incubated 20 min at 136 65?C to inactivate EcoRI. A 40 ?L aliquot of the sample was taken here and used as the digested control. The remaining sample was then diluted to 4 mL with ligation buffer [final concentrations were 1% Triton X-100, 1X T4 DNA Ligase React ion Buffer (NEB)] and incubated at 37?C for 30 min. After the addition of 4800 U of T4 DNA Ligase (NEB), each sample was incubated at 16?C O/N. Proteinase K was added to all samples including the controls at a final concentration of 65 ng/mL. Samples were then incubated at 65?C O/N to reverse the crosslinking. Af ter de-crosslinking, samples were combined with 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 4 min at 13,000 rpm. The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and subsequently diluted with 1 vol of dH2O. The sample was combined with 0.1 vol of 3M NaOAc pH=5.2 and 2.5 vol of 100% ethanol. Af ter incubating 1 hr at -80?C, samples were centrifuged 20 min at 4?C at13,000 rpm. Pellets were washed with 75% ethanol and centrifuged 5 min at 4? at13,000 rpm. Pe llets were air dried at R.T. prior to resuspension in an appropriate volume of TE. Loading adjustment and digestion efficiency tests were performed using previously described methods (Hagege et al. 2007). Loading adjustment was performed by SYBR green quantitative PCR to the yellow locus, and samples were adjusted accordingly before TaqMan quantitative PCR for 3C. Preparation of the Control Template for 3C To prepare a control template containing all possible ligation products, equimolar amounts of bacterial artificial chromosomes [RP48-36F20 & CH321-96A10 (CHORI)] 137 spanning the loci of interest were digested in 1X NEBuffer 3 with EcoRI (NEB) at a concentration of 12 U/?g DNA. Digest ed DNA was purified by phenol:chloroform extraction and ethanol precipitation. DNA was then ligated with T4 DNA ligase in 1X T4 DNA Ligase Reaction Buffer (NEB) at 16?C O/N. Ligated DNA was purified by phenol:chloroform extraction and ethanol precipitation. A second digest was performed using HindIII (NEB) to linearize any DNA circles. Digested DNA was purified by phenol:chloroform extraction and ethanol precipitation. Quantitative PCR for 3C Primers were designed to flank all EcoRI restriction sites within the region of interest. Custom TaqMan TAMRA Probes (Applied Biosystems) were designed with 5?FAM reporter dye and 3?TAMRA quencher dye. Real -time PCR reactions were prepared using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the recommended protocol. Each reaction was performed in quadruplicate. To normalize for the PCR efficiency of each primer pair/probe combination, the BAC control template was used to generate standard curves for each combination. Interaction frequencies were calculated based on the Ct values of each sample relative to the standard curve for the given primer pair/probe combination. Primers and probes used are listed in Table 3-3. 138 Table 3-1. Sources of data for tiling arrays, endo-siRNA, and genome features. Antibody or feature type Lab Year Cell Type Label in heatmap reference Source Peak calling1 Active promoters Multiple 2010 S2 S2: active promoters Negre et al. 2010, modMine GSM409457 , http://intermi ne.modencod e.org 7 AGO2 9D6 all 2011 all All 9D6 AGO2 this study GSE22623 6 AGO2 9D6 Lei 2011 S2 S2: 9D6 AGO2 this study GSE22623 2 AGO2 9D6 Lei 2011 S3 S3: 9D6 AGO2 this study GSE22623 2 AGO2 Mueller Lei 2011 S2 S2: Mueller AGO2 this study GSE22623 2 AGO2 9D6 without GAF Lei 2011 S2 S2: 9D6 AGO2 without GAF this study GSE22623 1 all-cisNAT Lai 2010 all 3' cis-NATs Okamura et al. 2008 Table S1 1 ASH1- mono Pirrotta 2010 Sg4 Schwartz (2010) Sg4: ASH1-mono Schwartz et al. 2010 GSM454525 3 ASH1-poly Pirrotta 2010 Sg4 Schwartz (2010) Sg4: ASH1-poly Schwartz et al. 2010 GSM454524 3 BEAF-32 Corces 2009 Kc Bushey (2009) Kc: BEAF-32 Bushey et al. 2009 Supplementa l files 1 BEAF-32 Corces 2009 Mbn2 Bushey (2009) Mbn2: BEAF-32 Bushey et al. 2009 Supplementa l files 1 BEAF-32 White 2010 embryo Negre (2010) embryo: BEAF-32 Negre et al. 2010 GSM409067 1 CP190 Corces 2009 Kc Bushey (2009) Kc: CP190 Bushey et al. 2009 Supplementa l files 1 CP190 Corces 2009 Mbn2 Bushey (2009) Mbn2: CP190 Bushey et al. 2009 Supplementa l files 1 CP190 White 2010 embryo Negre (2010) embryo: CP190 Negre et al. 2010 GSM409068 1 CTCF Corces 2009 Kc Bushey (2009) Kc: CTCF Bushey et al. 2009 Supplementa l files 1 CTCF Corces 2009 Mbn2 Bushey (2009) Mbn2: CTCF Bushey et al. 2009 Supplementa l files 1 CTCF White 2010 Kc Negre (2010) Kc: CTCF Negre et al. 2010 GSM409079 1 CTCF White 2010 S2 Negre (2010) S2: CTCF Negre et al. 2010 GSM409078 1 CTCF-C White 2010 embryo Negre (2010) embryo: CTCF-C Negre et al. 2010 GSM409069 1 CTCF-N White 2010 embryo Negre (2010) embryo: CTCF-N Negre et al. 2010 GSM409070 1 dSfmbt Mueller 2008 imaginal Oktaba (2008) imaginal: Sfmbt Oktaba et al. 2008 Table S3 3 DSP1 Cavalli 2009 embryo Schuettengruber (2009) embryo: Dsp1 Schuettengruber et al. 2009 Table S17.xls 1 E(z) Pirrotta 2006 Sg4 Schwartz (2006) Sg4: E(z) Schwartz et al. 2006 MEXP-535 3 GAF Cavalli 2009 embryo Schuettengruber (2009) embryo: GAF Schuettengruber et al. 2009 Table S17.xls 1 GAF Gilmour 2008 S2 Lee (2008) S2: GAF Lee Mol Cell Biol 2008 Supplementa l files 4 GAF White 2010 embryo Negre (2010) embryo: GAF Negre et al. 2010 GSM409071 1 H3K27Ac- F Pirrotta 2010 Sg4 Schwartz (2006) Sg4: H3K27Ac Schwartz et al. 2010 GSM454533 3 H3K27me3 Cavalli 2009 embryo Schuettengruber (2009) embryo: H3K27me3 Schuettengruber et al. 2009 Table S17.xls 1 H3K27me3 Pirrotta 2006 Sg4 Schwartz (2006) Sg4: H3K27me3 Schwartz et al. 2006 MEXP-535 3 H3K4me3 Cavalli 2009 embryo Schuettengruber (2009) embryo: H3K4me3 Schuettengruber et al. 2009 Table S17.xls 1 H3K4me3 Pirrotta 2010 Sg4 Schwartz (2010) Sg4: Schwartz et al. GSM454526 3 139 H3K4me3 2010 H3K4me3 White 2010 embryo Negre (2010) embryo: H3K4me3 Negre et al. 2010 GSM409075 1 H3K4me3 White 2010 Kc Negre (2010) Kc: H3K4me3 Negre et al. 2010 GSM409458 1 H3K4me3 White 2010 S2 Negre (2010) S2: H3K4me3 Negre et al. 2010 GSM409457 1 H3K9Ac Pirrotta 2010 Sg4 Schwartz (2010) Sg4: H3K9Ac Schwartz et al. 2010 GSM454529 3 Inactive promoters Multiple 2010 S2 S2: inactive promoters Negre et al. 2010, modMine GSM409457 , http://intermi ne.modencod e.org 7 insulator all all all insulators multiple, see methods see methods 6 mod(mdg4) White 2010 embryo Negre (2010) embryo: Mod(mdg4) Negre et al. 2010 GSM409072 1 NELF-B Gilmour 2008 S2 Lee (2008) S2: NELF-B Lee et al. 2008 Supplementa l files 4 NELF-E Gilmour 2008 S2 Lee (2008) S2: NELF-E Lee et al. 2008 Supplementa l files 4 PC Cavalli 2009 embryo Schuettengruber (2009) embryo: Pc Schuettengruber et al. 2009 Table S17.xls 1 PC Pirrotta 2006 Sg4 Schwartz (2006) Sg4: Pc Schwartz et al. 2006 MEXP-535 3 pcg all all all PcG multiple, see methods see methods 6 PH Cavalli 2009 embryo Schuettengruber (2009) embryo: Ph Schuettengruber et al. 2009 Table S17.xls 1 PHO Cavalli 2009 embryo Schuettengruber (2009) embryo: Pho Schuettengruber et al. 2009 Table S17.xls 1 PHO Mueller 2008 embryo Oktaba (2008) embryo: Pho Oktaba et al. 2008 Table S1 1 PHO Mueller 2008 imaginal Oktaba (2008) imaginal: Pho Oktaba et al. 2008 Table S2 1 PHOL Cavalli 2009 embryo Schuettengruber (2009) embryo: Phol Schuettengruber et al. 2009 Table S17.xls 1 PolII Pirrotta 2010 Sg4 Schwartz (2010) Sg4: PolII Schwartz et al. 2010 GSM454527 3 PolII White 2010 embryo Negre (2010) embryo: PolII Negre et al. 2010 GSM409077 1 PPEP Adelman 2007 S2 Muse (2007) S2: PPEP Muse et al. 2007 Table S1 1 PRE Muller 2008 imaginal Oktaba (2008) imaginal: PRE Oktaba et al. 2008 Table S4 1 PRE Pirrotta 2010 Sg4 Schwartz (2010) Sg4: PRE Schwartz et al. 2010 Table S6 1 promoters generated 2010 all FlyBase: 250bp upstream from TSS FlyBase FlyBase r5.29 1 PSC Pirrotta 2006 Sg4 Schwartz (2006) Sg4: Psc Schwartz et al. 2006 MEXP-535 3 redfly Gallo 2011 all Merged REDfly CRMs Gallo et al. 2011 http://redfly. ccr.buffalo.e du/ 1 siRNA- cluster Hannon- Siomi 2008 S2 S2: siRNA-clusters multiple, see methods see Methods 5 su(Hw) Corces 2009 Kc Bushey (2009) Kc: Su(Hw) Bushey et al. 2009 Supplementa l files 1 su(Hw) Corces 2009 Mbn2 Bushey (2009) Mbn2: Su(Hw) Bushey et al. 2009 Supplementa l files 1 su(Hw)-1 White 2010 embryo Negre (2010) embryo: Su(Hw)-Corces Negre et al. 2010 GSM409073 1 su(Hw)-2 White 2010 embryo Negre (2010) embryo: Su(Hw)-Geyer Negre et al. 2010 GSM409074 1 siRNA- overlapping 9D6 AGO2 sites This study 2011 S2 S2: siRNA-overlapping AGO2 9D6 sites Multiple, see methods See Methods 5 TRX-C Cavalli 2009 embryo Schuettengruber (2009) embryo: Trx-C Schuettengruber et al. 2009 Table S17.xls 1 140 TRX-C- Beisel Pirrotta 2010 Sg4 Schwartz (2010) Sg4: Trx-C-Beisel Schwartz et al. 2010 GSM454521 3 TRX-C- Poux Pirrotta 2010 Sg4 Schwartz (2010) Sg4: Trx-C-Poux Schwartz et al. 2010 GSM454521 3 TrxG all all all TrxG multiple, see methods see methods 6 TRX-N Pirrotta 2010 Sg4 Schwartz (2010) Sg4: Trx-N Schwartz et al. 2010 GSM454523 3 Description of original data sources used in this study including GEO or ArrayExpress accessions as available. 1Peak-calling methods performed in order to obtain BED-format files for this study 1 discrete features (peaks, domains, genes) available directly 2 MACS v1.3.7.1 FDR 5% 3 Distance-based clustering algorithm from (Negre et al., 2010) 4 WIG data segmented into peaks 5 see Supplemental Materials and Methods for siRNA analysis 6 Concatenated and merged from other BED files as described in Supplementary Materials and Methods 7 Identified using H3K4me3 and PolII 141 Table 3-2. List of primers. Primer Sequence Coordinates Application1 1F AGAAACCCATTGGTGCAGAC chr3R:12724311-12724330 CH 1R CAAAGTTGGATGCATTGTGG chr3R:12724422-12724441 CH 2F TCAAAGAGCGACACGTGAAC chr3R:12724828-12724847 CH 2R CATCAAACCTAGCCGCTCTC chr3R:12725015-12725034 CH 3F TCTTCGGGATGGCAATAAAC chr3R:12726187-12726206 CH 3R ACGATGTCGGATTCCTGAAC chr3R:12726318-12726337 CH 4F GGTTCTATTCCTAAAATTCTGTATGC chr3R:12728314-12728339 CH 4R GCATAACTCAAGGCCCGTTA chr3R:12728417-12728436 CH 5F CACGTGTTCGGTTTTCCTTT chr3R:12744456-12744475 CH 5R TTCCCTCCAATATGCAGACC chr3R:12744686-12744705 CH 6F GCAAGCGAAGAGTTCCATTC chr3R:12744868-12744887 CH 6R ACTGTCGGAGAGCGACATCT chr3R:12744997-12745016 CH 7F TGGTGGAAGGAGAAAACTGG chr3R:12746370-12746389 CH 7R TGCAGCGAGACAATAAAACG chr3R:12746602-12746621 CH 8F GCCAACCAGAAGGTCGTAAA chr3R:12749233-12749252 CH 8R GCTTCTCTTGGCGTTTCATC chr3R:12749340-12749359 CH 9F GCACTGTTTCAACTAGCGCCTTCA chr3R:1276037 1-12760394 CH 9R TTGAAGAGCGGATGCCTTCACACGTA chr3R:12760508-12760533 CH 10F CAAAGACGCGAACAAGTGAA chr3R:12770226-12770245 CH 10R TTGAACTTTGGCGGTACGAT chr3R:12770343-12770362 CH 11F CAAGTGAGTAGGCGATACGG chr3R:12774451-12774470 CH 11R CTCACGCTCTCGCAAAGTG chr3R:12774537-12774555 CH 12F CTCGTCTGCCTTCCATTCTC chr3R:12786264-12786283 CH 12R TTCTTTTGTCCGGGTAGTGG chr3R:12786350-12786369 CH 13F GGCGAATACGAAATCACCAC chr3R:12789485-12789504 CH 13R GTCAGGAGGAAACCACGAGA chr3R:12789567-12789586 CH 14F CACGCATTCTGCTGGTACAT chr3R:12795809-12795828 CH 14R CGGGCTCGTATCTGTGTCTC chr3R:12795961-12795980 CH RpL32F CTGCATGAGCAGGA chr3R:25871170-25871184 CH RpL32R ATGACCATCCGCCC chr3R:25871488-25871501 CH 17 CTATGGGCCGCCAAATACCGTTTC AA chrX:254644-254669 DE,LA 18 TGGAGACTACATTGCCTGAATTGGCG chrX:254797-254822 DE,LA 81 GGAATTTCTTTGTCATTTCCACTGTGCC chr3R:12743700- 12743727 DE,SP 82 CGCTCTCTATCTGATCATCACACACGGT chr3R:12743796-12743823 DE 83 ACAGAGTGCACTTGAGAAATCGGC chr3R:12744035-12744058 DE,SP 84 TTGAAATTCCCGGTTCAAGTGCGG chr3R:12744181-12744204 DE 85 CCGCACTTGAACCGGGAATTTCAA chr3R:12744181-12744204 DE,SP 86 TGAGACATCAGGAAGAGGTTCGTTGG chr3R:12744291-12744316 DE 87 TGTACATGAAGCATATGTAGACG chr3R:12745316-12745338 DE,SP,TRTP 88 CTTCTACTTGCAGAACTTGTACTC chr3R:12745394-12745417 DE 89 TTTCGTGGGTGGATGACTTTCCC chr3R:12746979-12747001 DE,SP,TRTP 90 GCAACATATGGTACTTCCTGCGCT chr3R:12747098-12747121 DE 91 TTTGAGCCAGAGGCTGACGACAT chr3R:12749676-12749698 DE,SP,TRTP 92 TCAGATGAGCAGAATGCCGAAGGA chr3R:12749770-12749793 DE 93 CATCCTCGGGCACTTTGAGACAACTT chr3R:12749899-12749924 DE,SP,TRTP 94 GATCTGCTGTTGGTTAGAACACCTTT chr3R:1274997 5-12750000 DE 103 AGTACGAAACACGAACTATGTGGGCG chr3R:12720571-12720596 DE,SP,TRTP 104 AAATCACGTTCGTCGGAAGTGGAGA chr3R:12720671-12720695 DE 105 AAGAGAGCGGCTAGGTTTGATGGT chr3R:12725013-12725036 DE,SP,TRTP 106 AGACTTGCCTCAGCCTCTGAAT chr3R:12725091-12725112 DE 107 ATTCAGAGGCTGAGGCAAGTCT chr3R:12725091-12725112 DE,SP 108 TCACCGCTAGAGTTGGAAACCAGT chr3R:12725211-12725234 DE 109 CCAACCATGCACACATCCAGGTAA chr3R:12729389-12729412 DE,SP,TRTP 110 TGAAGAGAAGGCGGTTGGTCTGTT chr3R:12729553-12729576 DE 111 TCATGTCGATTTCAGTCCGTAGCCAG chr3R:12730755-12730780 DE,SP 112 TTAGCCCTGCCATAAAGTTCGGTTCC chr3R:12730894-12730919 DE 113 TATGCAGTTGACGTCGGTTGATGC chr3R:12732700-12732723 DE,SP 114 ACGAGATGGTGCGTCCATAAAGGT chr3R:12732797-12732820 DE 115 TTGTTGTTCTGCTGATTGGCCTGG chr3R:12755226-12755249 DE,SP 116 TTGGCCAGAAATTTGCAGCTGACC chr3R:12755369-12755392 DE 117 GTCTTGGTAGCATTGAACAGTTAGGACAG chr3R:12755990-12756018 DE,SP 118 AGTTAAGTGACCTCGCCAGCCAAT chr3R:12756076-12756099 DE 119 ACATTTAGGTGGAATTTGAACGCCTCT chr3R:12756585-12756611 DE,SP,TRTP 120 GCATCTTGCAACTCTAGTTTGGGAGG chr3R:12756742-12756767 DE 142 121 GCACTGTTTCAACTAGCGCCTTCA chr3R:12760371-12760394 DE,SP,TRTP 122 TTGAAGAGCGGATGCCTTCACACGTA chr3R:12760508-12760533 DE 123 CGCATTTAGTTGAAGAGTCCAACTGCT chr3R:12761292-12761318 DE,SP,TRTP 124 GGAAATAGATTGCGGCAGTTAATTACAAGT chr3R:12761385-12761414 DE 125 GAATGGGAAAAGTTTCCGGCCTAAC chr3R:12738565-12738589 DE,SP,TRTP 126 GGAAACATATTTTGGGATGGGCTTT chr3R:12738732- 12738756 DE 127 TTCGCCGCCATTTGCCGAAGG chr3R:12746942-12746962 DE,SP,TRTP 129 AGAGGTAGTTAGACGATCGTGGGT chr3R:12768301-12768324 DE,SP,TRTP 130 TGAGTGGATTTGACCACTTGGGTG chr3R:12768412-12768435 DE 131 ATTCTGGCGATTCTGTCCCTTCCA chr3R:12782513-12782536 DE,SP,TRTP 132 CTGGCATAGCAACGTAACAACTATGGG chr3R:12782635-12782661 DE 133 ACCGACATCTTCATATCTGCCTTGC chr3R:12782989-12783013 DE,SP 134 CGAAATTAAAGCATGTTCTCATTTAGG chr3R:12783106-12783132 DE 135 CCTAAATGAGAACATGCTTTAATTTCGG chr3R:12783106- 12783133 DE,SP 136 GCTCGAAAAATCCAAGATAATTGACTGACC chr3R:12783215-12783244 DE 137 GCACTCTCATATTTCCAAGAGCACACC chr3R:12784025- 12784051 DE,SP,TRTP 138 GGGTGTGTCCATACTTGCACTGT chr3R:12784180-12784202 DE 139 CTCTCGTGGTTTCCTCCTGACC chr3R:12789566-12789587 DE,SP,TRTP 140 TGTGTGTGTCAGGTGTTGTTACCC chr3R:12789734-12789757 DE 141 AGAAGGACAAGGGAATGGGTGTGA chr3R:12790763-12790786 DE,SP,TRTP 142 AGCAACTGCGGAGGCCATAAATTG chr3R:12790879-12790902 DE 143 TCAATTGAAGCGCATCGCAACCGT chr3R:12791097-12791120 DE,SP,TRTP 144 AGAAGCATGCTCCAGTTGACCCAA chr3R:12791184-12791207 DE 145 CAAATATGCCGCCGGCTTTGGAAT chr3R:12801377-12801400 DE,SP,TRTP 146 GGAGCTGCAAGGCTATCTTGATATGTATG chr3R:12801639-12801667 DE 147 AAGATAGGAGTGGATGATGGCGCA chr3R:12801832-12801855 DE,SP,TRTP 148 TTCGGAGATCGACGTTTAAGCCTG chr3R:12801940-12801963 DE 121Probe TCCAGAGCCAGTCCCAGTCGAAGTG chr3R:12760415-12760439 TRTP 89Probe TCTCAGCACGCGCTTTTCGTGG chr3R:127469650-12746986 TRTP 125Probe TGCGAGTTTATTAACCGCAAGTAATTT CACCAAA chr3R:12738596-12738629 TRTP AGO2-S caaccacagcagcugcaacdTdT chr3L:15547468-15547490 SI AGO2-AS guugcagcugcugugguugdTdT chr3L:15547571-15547593 SI Pc Fwd TAATACGACTCACTATAGGGAGAGTACCGTGTCAAGTGGAAG chr3L:21309750-21310571 DS Pc Rev TAATACGACTCACTATAGGGAGATTTGGTATGTTATTGTTCTCGG DS TRX Fwd TAATACGACTCACTATAGGGAGACAAACGTCTACCACCACCC chr3R:10098104-10098949 DS TRX Rev TAATACGACTCACTATAGGGAGAGCCCATTAGCGTGGTATCC DS 1Legend CH Chromatin Immunoprecipitation DS dsRNA Amplicons DE Digestion Efficiency Test LA Loading Adjustment Test SP Standard PCR Detection of Interactions TRTP TaqMan Real-time PCR Detection of Interactions SI siRNA knockdowns 143 CHAPTER 4 DISCUSSION AND FUTURE DIRECTIONS RNA SILENCING AND ITS ROLE IN HETEROCHROMATIN ASSEMBLY piRNA- and endo-siRNA-mediated TE silencing Our results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways. These findings suggest two possibilities for the heterochromatic silencing of TEs; sites other than piRNA producing loci serve as Piwi-dependent HP1 recruitment sites in Drosophila genome, or the existence of mechanisms alternative to RNA silencing that recruit HP1 to chromatin. We considered the first possibility and, in order to gain insight into the genome-wide chromatin association of Piwi, performed ChIP-seq of Piwi in Drosophila ovarian somatic cell (OSC) line. Our preliminary analysis revealed Piwi association with repeat-rich sequences but not with euchromatin (data not shown) indicating that no other Piwi-dependent HP1 binding platforms exist in euchromatin. Interestingly, our directed HP1 ChIP detected a two-fold decrease in HP1 recruitment at the 1360 element in OSC cells depleted of Piwi when compared to mock- treated cells. Coincidently, the 1360 and F elements, two TEs known to be preferentially bound by HP1, were shown to bind Piwi (reviewed in Brower-Toland et al. 2007). Whether HP1 recruitment to the F element is affected in Piwi-depleted OSC cells is 144 unknown. The same experiment, however, revealed that an additional target of HP1, TART, a telomere-specific non-LTR retrotransposon, was immunoprecipitated with HP1 at levels similar between mock-treated and Piwi-depleted OSC cells. Therefore, it is possible that a differential mechanism of TE heterochromatic silencing that may depend on Piwi exists at specific genomic sites. Further analysis will reveal the identity of these Piwi-associated repeat-rich sequences. It is likely that different Piwi clade proteins silence TEs by mechanisms that may involve additional proteins. A recent study reported that one HP1 variant, Rhino, may play a role in piRNA-mediated TE silencing (Klattenhoff et al. 2009). Rhino, which is specifically expressed in the female germline, is required for TE silencing, and its localization to nuclear foci is independent of piRNA production. A model, which is mechanistically distinct from centromeric heterochromatin silencing in yeast, was proposed where Rhino does not appear to be involved in TE silencing. According to this model, Rhino binds to 42AB piRNA cluster to promote tr anscription of piRNAs, which associate with Aub and AGO3, that most likely direct TE silencing through posttranscriptional target cleavage. The authors speculated that rhino, which is a rapidly evolving gene, may be involved in a battle between TE propagation and maintenance of germline DNA integrity. The TE integration machinery is constantly evolving to escape silencing. Rhino?s rapid evolution may be due to interaction with TE integration proteins in order to promote transposition into the piRNA clusters and generate trans-silencing piRNAs. It has been hypothesized that piRNA clusters serve as hot spots for TE entrapment but the mechanistic details of this phenomenon and the role that HP1 variants may play have not been elucidated. 145 Our results also demonstrate no requirement for AGO2 in HP1 recruitment in somatic tissues. Furthermore, high-resolution genome-wide chromatin association profile of AGO2 in S2 and S3 Drosophila embryonic cell lines revealed that chromatin- associated AGO2 localizes in euchromatic but not repeat-rich regions. When compared to regions of the genome that produce Dicer-dependent endo-siRNAs, AGO2 genome- wide localization revealed no overlap. We also performed AGO2 ChIP-seq in OSC line which expresses AGO2 at high levels. Our preliminary results revealed that the majority of chromatin-associated AGO2 localizes in euchromatic regions. A more in depth computational analysis will reveal whether there is any AGO2 enrichment for repetitive sequences in ovarian somatic cells. Alternative mechanisms for heterochromatin nucleation Our results show that piRNA and endo-siRNA pathways do not recruit heterochromatin to the piRNA producing loci in Drosophila somatic tissues. We also show that HP1 recruitment to chromatin is independent of Piwi in the ovarian somatic cells. Studies have shown that heterochromatin formation can be achieved independently of RNAi. In fission yeast, the ATF/CREB stress-activated proteins nucleate heterochromatin at the silent mating-type locus in an RNAi-independent manner (Jia et al. 2004). Also, fission yeast telomere binding protein Taz1 can establish HP1 recruitment to telomeres independent of RNAi (Kanoh et al. 2005). In Drosophila , DDP1 dodeca-satellite binding protein, a single-stranded nucleic acid binding protein that associates with pericentric heterochromatin, has been suggested to contribute to 146 heterochromatin organization and function (Cortes and Azorin 2000). Furthermore, a recent study in mouse cells demonstrated that long nuclear non-coding transcripts that correspond to major satellite repeats at the pericentric heterochromatin associate with SUMO-modified HP1, a modification that promotes the initial targeting of HP1 to these regions (Maison et al. 2011). The same study did not detect any small dsRNA corresponding to major satellites suggesting that RNAi-mediated heterochromatin assembly, as pertains to S. pombe, may be a pathway that is not evolutionary conserved in metazoans. RNA SILENCING AND ITS EFFECTS ON CHROMATIN INSULATORS AGO2: a multifunctional protein Our findings suggest two distinct roles for AGO2. In addition to its RNAi- dependent posttranscriptional silencing of TEs, we show that AGO2 functions in euchromatin in a Dicer-independent manner to promote or stabilize CTCF/CP190- dependent looping interactions that define transcriptional domains throughout the genome. Although, we show that the Slicer activity of AGO2 is not required for Fab-8 activity and CP190 mutants, which lose AGO2 association with chromatin, are still functional for RNAi, whether the functions of AGO2 in RNAi and chromatin insulator activity are completely separate remains to be elucidated. 147 Functionally multifaceted proteins are not a rare phenomenon in eukaryotic systems. AGO2 localizes to insulators, PREs, and promoters. One common feature of these sites is that they correspond to high nucleosome turnover, presumably needed to permit access to the macromolecular machinery that defines their activities (Mito et al. 2007; Deal et al. 2010). Intriguingly, de novo motif analysis of AGO2 binding sites resulted in a GA-rich motif similar to the GAF binding sequence. As multifunctional as AGO2, GAF is required for Fab-7 and SF1 insulator activitie s (Belozerov et al. 2003; Schweinsberg et al. 2004), has been classified as a trxG protein (Farkas et al. 1994), and is associated with NELF-dependent paused polymerases (Lee et al. 2008). GAF associates with DNA directly through a zinc-finger DNA-binding domain, which is not present in AGO2 (reviewed in Adkins et al. 2006). However, both proteins harbor a polyglutamine-rich region of unknown function, which could mediate interactions with common proteins. Since AGO2 binding sites that do not overlap with GAF still contain the GA-rich motif, GAF binding does not appear to be prerequisite for AGO2 binding. Furthermore, in CP190 mutants, AGO2 but not GAF chromatin association is lost, suggesting that GAF and AGO2 recruitment are achieved by independent mechanisms. Over twenty years of genetic and biochemical studies suggests that GAF promotes open chromatin, although, the mechanism is not well understood. We propose that once recruited to chromatin by CP190 and CTCF, AGO2 could serve to open chromatin and promote insulator activity by maintaining a nucleosome-free state and stabilizing or increasing the frequency of looping interactions between insulators, promoters, and PREs. It has been proposed that GAF may recruit to nucleosome remodeling factors to assist in local nucleosome turnover, which depending on gene 148 context, could lead to transcriptional activation or suppression (Xiao et al. 2001). Identification of additional proteins interacting with AGO2 such as components of the chromatin remodeling machinery may shed more light on the mechanism of AGO2 associated with chromatin. Role for AGO2 in transcriptional regulation The observation that more than half of AGO2 sites are associated with promoters suggests that AGO2 may regulate transcription directly. Given that AGO2 behaves similarly to a trxG protein, we anticipated that AGO2 may activate transcription. Transcription per se, however, may not be required for AGO2 chromatin association since TrxG proteins, depletion of which reduces transcription of Abd-B (Schwartz et al. 2010), are not required for AGO2 recruitment to chromatin. Interestingly, AGO2 depletion leads to either upregulation or downregulation of hundreds of transcripts (Rehwinkel et al. 2006). Upregul ation would be consistent with direct posttranscriptional regulation via the RNAi pathway, but these effects could alternatively be due to direct transcriptional repression. Despite potentially complex relationships between AGO2 and gene expression levels, we found that the promoters of a statistically significant number of upregulated transcripts are bound by AGO2 (data not shown), suggesting that AGO2 may negatively regulate some of the transcripts to which it binds. It is unclear why some transcripts, whether bound by AGO2 or not, are downregulated upon AGO2 depletion. These gene expression changes could indicate an additional function for AGO2 in transcriptional activation that will be elucidated by future research. Alternatively, these 149 events could result from secondary effects of increased expression of RNAi-dependent or transcriptional targets of AGO2. We also observed a considerable genome-wide overlap between AGO2, paused Pol II, and NELF, which is required for promoter proximal pausing of Pol II. Despite the overlap, AGO2-dependent gene expression does not correlate with NELF-dependent transcriptional effects. It was shown that depletion of NELF leads to both positive and negative effects on gene expression via two independent mechanisms (Muse et al. 2007; Gilchrist et al. 2008). On the one hand, NELF can attenuate gene expression by maintaining Pol II in a paused state in cases where transcription elongation is rate- limiting. On the other hand, NELF also promotes gene expression by stabilizing paused Pol II and maintaining an open chromatin structure at the promoter. Because promoters with paused polymerase, including Abd-B RB, have recently been shown to possess NELF-dependent enhancer blocking activity in transgene assays (Chopra et al. 2009), we compared gene expression profiles of AGO2 or NELF depletion in S2 cells but did not identify any statistically significant correlation (data not shown). We also compared the AGO2-dependent transcription profile with that of CP190 or CTCF but did not detect any resemblance (data not shown). Like AGO2, extensive promoter association has been reported for the insulator proteins CP190, CTCF, Mod(mdg4)2.2, and BEAF-32 but not Su(Hw) (Bushey et al. 2009; Jiang et al. 2009; Smith et al. 2009; Negre et al. 2010). In CP190 and CTCF depleted S2 cells, gene expression changes are also observed in both positive and negative directions (Bartkuhn et al. 2009). Correlation between binding and change in gene expression was identified for both positively and negatively regulated transcripts, with CP190 more frequently 150 found at the promoter than CTCF. Our correlation analysis of gene expression changes among AGO2, CP190, or CTCF depleted cells indicates that there is no statistically significant correlation between AGO2 and either insulator protein. Currently, the significance of AGO2 or insulator protein promoter association is unclear. A connection between two functions of AGO2 AGO2 may be an example of how the cell uses the same protein for two distinct purposes since the differential effects of the AGO2 loss-of-function mutant and AGO2 catalytic activity mutant on Fab-8 insulator activity and Pc suppression suggests that AGO2 function in chromatin organization may be uncoupled from its role in RNAi. In order to understand whether the functions of AGO2 in RNAi and chromatin can be separated a systematic dissection of different aspects of AGO2 structure need to be performed. We show that AGO2, present in the nuclear pool, interacts with CP190 and CTCF in an RNA-independent manner. However, whether AGO2 can interact with siRNAs while associated with the insulator complex is not clear. Although, no small RNAs associated with the insulator proteins could be identified (data not shown), examining AGO2 mutants defective in the PAZ and Mid domains, which would lack the ability to bind small RNAs, on Fab-8 insulator activity and Pc suppression may address that question. The regulation of RNAi and chromatin-related activities may be directed by AGO2 present in the cytoplasmic and nuclear fractions, respectively. An emerging theme from studies of post-translational modifications of mammalian AGO proteins is 151 that these modifications are crucial for the function of AGO proteins. One study identified hydroxylation of the human endogenous Ago2, a modification that appears to be crucial for stability of Ago2 and proper function of RISC activity (Qi et al. 2008). Another study identified phosphorylation of human Ago2, which contributes to Ago2 localization (Zeng et al. 2008). Also, a study in mice reveal ed that ubiquitylation of Ago2 affects its turnover (Rybak et al. 2009). It would be interesting to see whether the functions of AGO2 in RNAi and chromatin may be modulated by distinct post- translational modifications. Future research will examine Drosophila AGO2 modifications in the context of transcriptional regulation by two different mechanisms. Another possibility is that distinct AGO2 isoforms may influence RNAi and chromatin organization in a different manner. The AGO2 locus is predicted to give rise to two transcripts, AGO2-RB and AGO2-RC (FlyBase, 6_2010). The two isoforms are mostly identical except for distinct transcription start sites due to alternative splicing. The two transcripts encode two protein isoforms that differ only by 6-9 amino acids at their amino termini and have a 1208 amino acid region in common. A recent study in Drosophila reported that the AGO2 locus produces an alternative transcript, which is predicted to encode a putative short isoform of AGO2 that is lacking the amino-terminal domain (Hain et al. 2010). However, the authors were not able to verify protein production from this transcript in vivo. Future studies will address whether different AGO2 isoforms vary in abundance in cytoplasmic and nuclear pools and whether they play distinct roles in these cellular compartments. Lastly, AGO2 loss-of-function mutant and AGO2 catalytic activity mutant may regulate transcription of different target genes. 152 Therefore, performing a gene expression profiling of these mutants in order to identify their targets may also be of interest. AGO2 role in nuclear organization Nuclear bodies such as insulator and PcG bodies have been proposed to be hubs of nuclear proteins interacting with regulatory elements in order to organize chromatin. Previously it was shown that changes in gypsy insulator activity in piwi and aub mutants corresponds to an improvement in the overall organization of insulator bodies (Lei and Corces, 2006), a structure that may be es tablished in the early embryo and persist throughout development. Since no changes in insulator protein expression or recruitment to chromatin have been detected in these mutants, these RNA silencing pathways appear to specifically affect nuclear organization of the gypsy insulator. We examined gypsy insulator activity in AGO2 mutants and found it to be similarly improved (data not shown). An increase of gypsy insulator activity in AGO2 mutants corresponds to an improvement in the overall organization of insulator bodies. Moreover, localization of gypsy insulator proteins Su(Hw) and CP190 in Drosophila polytene chromosomes is not altered in AGO2 mutants (data not shown). Interestingly, neither the ability of CTCF and CP190 to associate with chromatin or specifically with the BX-C nor localization of insulator bodies is affected in AGO2 mutants. Therefore, it appears that Fab-8 function is uncoupled from insulator body formation since Fab-8 activity is not affected by RNA silencing mutants that alter insulator body localization. These observations suggest a differential effect of AGO2 on different classes of insulators. It would be interesting to 153 examine AGO2 effects on other classes of Drosophila insulators such BEAF-bound scs? insulator and ZW5-defined scs insulator. Given that the two insulators have been shown to interact by looping out the intervening DNA (Blanton et al. 2003), investigating the effects of AGO2 on this communication may also be worth of attention. Unlike RNA silencing mutants, ago1, piwi and aub, which disrupt PcG-mediated long-range interactions between PREs, AGO2 does not appear to function in this phenomenon. Although, we did not determine whether AGO2 localizes to PcG bodies, a mutation in AGO2 has no effect on Fab-X. Future studies will elucidate the role of AGO2 in Mcp PRE-mediated long-range interactions. Overall, however, our findings indicate that AGO2 function in general chromatin organization may not necessarily be connected to the formation of nuclear bodies. CONCLUSION Here, we demonstrate that RNA silencing affects gene expression at the level of higher order chromatin organization in Drosophila melanogaster. Specifically, our findings reveal that small RNA silencing pathways do not mediate heterochromatin formation in fly somatic tissues suggesting that alternative mechanisms may be involved. We also uncover a novel Dicer-independent function of AGO2 in CTCF/CP190 insulator activity. The role of AGO2 in chromatin organization is intriguing but whether it is conserved in mammals remains an open question. Having a highly divergent sequence, 154 Drosophila AGO2 is very much distinct from AGO1, as well as yeast, plant, and mammalian Argonautes. Its carboxy-terminal region is conserved from plants to vertebrates, and the PAZ and PIWI domains are well characterized for RNA binding and cleavage. The AGO2 amino-terminal region is uniquely long in comparison to other Argonautes. This domain includes long stretches of glutamine-rich repeats, and its function is not well understood (Meyer et al. 2006). Future studies will address which domain of Drosophila AGO2 is responsible for its association with chromatin. It remains an open question whether Argonaute function in chromatin is conserved in the mammalian CTCF activity as well as long-range chromosomal interactions mediated by this insulator protein. Additionally, two recent studies identified an endogenous PRE in the human homeotic genes HOXD12 and HOXD11 and a mouse PRE (Sing et al. 2009; Woo et al. 2010). Interestingly, the mouse PRE contains GAGA- binding motif. Future research will address whether AGO2 binding to PREs is a conserved phenomenon. Our findings provide a first glimpse into understanding the complex interplay between the RNA silencing machinery and higher order chromatin structure to affect changes in gene expression. 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