CONTROL AND FUNCTION OF HISTONE H3 LYSINE 27 METHYLATION IN NEUROSPORA CRASSA by KEVIN JOSEPH MCNAUGHT A DISSERTATION Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2019 DISSERTATION APPROVAL PAGE Student: Kevin Joseph McNaught Title: Control and Function of Histone H3 Lysine 27 Methylation in Neurospora crassa This dissertation has been accepted and approved in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Biology by: Dr. Jeffrey McKnight Chairperson Dr. Eric Selker Advisor Dr. Alice Barkan Core Member Dr. Kryn Stankunas Core Member Dr. Michael Harms Institutional Representative and Kate Mondloch Interim Vice Provost and Dean of the Graduate School Original approval signatures are on file with the University of Oregon Graduate School. Degree awarded December 2019 ii © 2019 Kevin Joseph McNaught iii DISSERTATION ABSTRACT Kevin Joseph McNaught Doctor of Philosophy Department of Biology December 2019 Title: Control and Function of Histone H3 Lysine 27 Methylation in Neurospora crassa In higher eukaryotes, proper development and maintenance of cellular identity requires the activity of Polycomb repressive complexes (PRCs). PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), which serves as a repressive chromatin mark. PRC2 is conserved in the filamentous fungus, Neurospora crassa, and H3K27 methylation covers around 7% of the genome, repressing scores of genes. My research focused on two basic questions: 1. What controls the distribution of H3K27 methylation? and 2. How does H3K27 methylation function as a repressive mark? As one approach to identify factors that influence the deposition of H3K27 methylation, I screened gene knock-outs of nst (neurospora sir two) homologues for defects in H3K27 methylation. I discovered that loss of NST-3, a putative H3K56 deacetylase, results in accumulation of H3K27 methylation at centromeres, and that this is suppressed by strains lacking rtt109 or bearing histones with the H3K56R substitution. In collaboration with Kirsty Jamieson, we investigated what role chromosome position had on the deposition of H3K27 methylation by examining chromosomal rearrangement strains. We identified both position-dependent and position-independent H3K27 methylation. We further found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive H3K27 methylation, and iv that this is effect is mediated by telomere repeats, (TTAGGG)n. As a part of a forward-genetics selection for factors necessary for Polycomb silencing, spearheaded by Elizabeth Wiles, I identified two alleles of a gene, NCU04278, that is required for the telomere-dependent H3K27 methylation uncovered in our previous work. Immunoprecipitation followed by mass spectrometry analyses of NCU04278 and known PRC2 members established that NCU04278 is a PRC2 accessory subunit and was therefore designated PAS. The aforementioned forward genetic selection identified another gene, epr-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27 methylation in vivo and in vitro, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR- 1 was a component of a primitive Polycomb repression pathway. This dissertation includes both previously published and unpublished co-authored material. v CURRICULUM VITAE NAME OF AUTHOR: Kevin Joseph McNaught GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene DEGREES AWARDED: Doctor of Philosophy, Biology, 2019, University of Oregon Bachelor of Science, Biology, 2012, University of Oregon AREAS OF SPECIAL INTEREST: Epigenetics Chromatin Genomics PROFESSIONAL EXPERIENCE: Graduate Student, Eric Selker laboratory, University of Oregon, 2013-2019 Teaching Fellow, Department of Biology, University of Oregon, 2013-2014 GRANTS, AWARDS, AND HONORS: Developmental Biology Training Grant, National Institutes of Health, 2015-2018 PUBLICATIONS: Jamieson, K.*, McNaught, K.J.*, Ormsby, T., Leggett, N.A., Honda, S., and Selker, E.U. (2018). Telomere repeats induce domains of H3K27 methylation in Neurospora. eLife 7, 948. Jamieson, K., Wiles, E.T., McNaught, K.J., Sidoli, S., Leggett, N., Shao, Y., Garcia, B.A., and Selker, E.U. (2016). Loss of HP1 causes depletion of H3K27me3 from facultative heterochromatin and gain of H3K27me2 at constitutive heterochromatin. Genome Research 26, 97–107. *These authors contributed equally to this work. vi ACKNOWLEDGMENTS I’d like to thank Eric Selker for his continued support and guidance throughout my graduate career. My lab mates, Kirsty Jamieson and Elizabeth Wiles, were incomparable collaborators and provided much valuable feedback. In addition, I owe a debt to all previous and current Selker lab members, who paved the way for my success. I also want to thank the members of my dissertation committee for their encouragement and advice, and the Fungal Genetics Stock Center for providing valuable Neurospora crassa strains. This investigation was supported in part by a National Institutes of Health Developmental Biology Training Grant. vii I dedicate this dissertation to my parents, Joe and Marybeth, for their unwavering love and support. viii TABLE OF CONTENTS Chapter Page I. INTRODUCTION .................................................................................................... 1 II. IMPLICATION OF ABERRANT H3K56 ACETYLATION IN ECTOPIC H3K27 METHYLATION ........................................................................................................ 4 Introduction ............................................................................................................ 4 Results .................................................................................................................... 5 Deletion of nst-3 results in centromeric H3K27 methylation .......................... 5 Centromeric H3K27 methylation suppressed in ∆nst-3 and ∆hpo strains lacking H3K56 acetylation .............................................................................. 6 H3K56 acetylation appears to accumulate at centromeres in ∆nst-3 strains ... 7 Discussion .............................................................................................................. 8 Materials and Methods ........................................................................................... 9 Strains, media and growth conditions .............................................................. 9 Chromatin immunoprecipitation (ChIP) and ChIP-sequencing ....................... 9 Replacement of endogenous histone H3 with H3K56R/Q mutants ................. 9 Bridge to Chapter III .............................................................................................. 10 III. TELOMERE REPEATS INDUCE DOMAINS OF H3K27 METHYLATION IN NEUROSPORA ...................................................................................................... 11 Introduction ............................................................................................................ 11 Results .................................................................................................................... 13 Analyses of classical chromosome rearrangements suggest that proximity to a chromosome end is key to subtelomeric H3K27 methylation .................. 13 ix Chapter Page Identification of position-independent H3K27-methylated domains .............. 15 Cis-trans test on H3K27 methylation in a segmental duplication strain reveals position effect ................................................................................................... 16 Altered gene expression in regions with changed H3K27me2/3 ..................... 17 Loss of telomerase abolishes subtelomeric H3K27me2/3 ............................... 18 Internal telomere repeats are sufficient to induce de novo H3K27me2/3 ........ 19 Discussion .............................................................................................................. 20 Materials and Methods ........................................................................................... 26 Strain, media and growth conditions ............................................................... 26 Deleting and targeting segments of LG VIL .................................................... 26 Deletion of tert ................................................................................................. 27 Targeting telomere repeats to csr-1 and his-3 .................................................. 27 ChIP and preparation of libraries ..................................................................... 28 RNA isolation and preparation of libraries ...................................................... 29 Translocation breakpoint analyses ................................................................... 29 Southern analysis ............................................................................................. 29 SNP mapping ................................................................................................... 30 Bridge to Chapter IV .............................................................................................. 30 IV. IDENTIFICATION OF A PRC2 ACCESSORY SUBUNIT REQUIRED FOR SUBTELOMERIC H3K27 METHYLATION IN NEUROSPORA ........................... 32 Introduction ............................................................................................................ 32 Results .................................................................................................................... 33 x Chapter Page Isolation, mapping and identification of NCU04278 as a potential PcG gene .......................................................................................................... 33 NCU04278 is necessary for silencing subtelomeric H3K27 -methylated genes ............................................................................................ 34 NCU04278 is necessary for maintenance of subtelomeric H3K27 methylation ...................................................................................................... 36 NCU04278 is an accessory subunit of PRC2 .................................................. 37 Discussion .............................................................................................................. 38 Materials and Methods ........................................................................................... 40 Strains, media and growth conditions .............................................................. 40 Selection for mutants defective in H3K27 methylation-mediated silencing ........................................................................................................... 40 Whole genome sequencing, mapping and identification of NCU04278 alleles .............................................................................................................. 40 Chromatin immunoprecipitation (ChIP), ChIP-qPCR and ChIP-seq .............. 41 RNA isolation, RT-qPCR and mRNA-seq ...................................................... 41 Immunoprecipitation followed by mass spectrometry (IP-MS) ...................... 41 Replacement of NCU04278 with trpC::nat-1 .................................................. 42 Generation of a N. crassa strain expressing NCU04278::3xFLAG ................ 43 Bridge to Chapter V ............................................................................................... 43 xi Chapter Page V. EVOLUTIONARILY ANCIENT BAH-PHD PROTEIN MEDIATES POLYCOMB SILENCING ................................................................................................................ 45 Introduction ............................................................................................................ 45 Results .................................................................................................................... 47 Genetic selection for factors necessary for H3K27 methylation-mediated repression ......................................................................................................... 47 Mapping and identification of epr-1 as NCU07505 ........................................ 48 EPR-1 and SET-7 repress an overlapping set of H3K27-methylated genes .... 49 ∆epr-1 and ∆set-7 strains share a defect in sexual development ..................... 50 H3K27 methylation is essentially normal in ∆epr-1 ........................................ 50 EPR-1 forms telomere-associated foci dependent on EED ............................. 52 An intact BAH domain is required for normal nuclear distribution of EPR-1 .............................................................................................................. 53 EPR-1 localizes to H3K27 methylation genome-wide .................................... 53 Both the BAH domain and PHD finger of EPR-1 are necessary for gene repression ......................................................................................................... 54 BAH domain of EPR-1 binds to H3K27me3 in vitro ...................................... 55 EPR-1 is a homolog of plant EBS/SHL and widely distributed across eukaryotes ........................................................................................................ 56 Discussion .............................................................................................................. 57 Materials and Methods ........................................................................................... 59 Strains, media and growth conditions .............................................................. 59 xii Chapter Page Selection for mutants defective in H3K27 methylation-mediated silencing ........................................................................................................... 60 Whole genome sequencing, mapping and identification of epr-1 alleles ........ 60 Western blotting ............................................................................................... 61 Chromatin immunoprecipitation ...................................................................... 62 ChIP-seq mapping and analysis ....................................................................... 63 RNA isolation, mRNA-seq library prep, and RT-qPCR .................................. 63 mRNA-seq mapping and analysis .................................................................... 64 DamID Southern hybridizations and sequencing ............................................ 64 False perithecia assay and image analysis ....................................................... 65 Microscopy image acquisition and analysis .................................................... 65 Cloning, expression and purification of BAH domain from EPR-1 ................ 66 NMR spectroscopy ........................................................................................... 67 Bioinformatic identification of EPR-1 and SET-7 homologs .......................... 68 Replacement of NCU05173 and NCU07152 ORFs with hph and nat-1 ......... 68 Creation and targeting of his-3+::pCCG::N-GFP::EPR-1 plasmids ................ 69 Replacement of epr-1 with trpC::nat-1 ............................................................ 70 Endogenous C-terminal-tagging of EPR-1 with 10xGly::Dam ....................... 70 VI. CONCLUDING SUMMARY ............................................................................... 72 APPENDICES ............................................................................................................. 76 A. FIGURES .......................................................................................................... 76 Chapter II .................................................................................................. 76 xiii Chapter Page Chapter III .................................................................................................. 77 Chapter IV .................................................................................................. 90 Chapter V .................................................................................................. 97 B. STRAIN TABLES ............................................................................................ 111 Chapter II .................................................................................................. 111 Chapter III .................................................................................................. 111 Chapter IV .................................................................................................. 112 Chapter V .................................................................................................. 113 C. PRIMER TABLES ............................................................................................ 115 Chapter II .................................................................................................. 115 Chapter III .................................................................................................. 115 Chapter IV .................................................................................................. 121 Chapter V .................................................................................................. 123 D. PLASMID TABLES ......................................................................................... 127 Chapter II .................................................................................................. 127 Chapter III .................................................................................................. 127 Chapter IV .................................................................................................. 127 Chapter V .................................................................................................. 127 REFERENCES CITED ................................................................................................ 129 xiv LIST OF FIGURES Figure Page 1. Aberrant H3K56ac implicated in ectopic H3K27me2/3 ........................................ 76 2. Chromosomal rearrangements are associated with altered H3K27me2/3 ............. 77 3. Three kilobase segments from a natural H3K27me2/3 domain are insufficient to trigger de novo H3K27me2/3 at ectopic loci ..................................................... 78 4. H3K27me2/3 profiles of additional strains containing chromosomal rearrangements ....................................................................................................... 79 5. Whole genome view of H3K27me2/3 ChIP-seq in wild-type and chromosomal translocation strains ............................................................................................... 80 6. qPCR validation of H3K27me2/3 ChIP-seq data from translocation strains ........ 81 7. A segmental duplication confirms H3K27me2/3 position effect .......................... 82 8. Altered gene expression reflects changes in H3K27me2/3 ................................... 83 9. qPCR validation of RNA-seq expression changes in translocation OY350 .......... 84 10. Loss of tert disrupts chromosome ends and abolishes subtelomeric H3K27me2/3 .......................................................................................................... 85 11. Whole genome view of H3K27me2/3 ChIP-seq in wild type and tert .................. 86 12. Telomere repeats targeted to the csr-1 locus induce an approximately 225 kb H3K27me2/3 domain ................................................................................. 87 13. Telomere repeats targeted to the his-3 locus induce local H3K27me2/3 domain .................................................................................................................... 88 14. Whole genome view of H3K27me2/3 ChIP-seq in wild type and Δcsr-1::(TTAGGG)17 ............................................................................................. 89 xv Figure Page 15. Identification of NCU04278 as a potential component of the Polycomb silencing pathway ................................................................................................... 90 16. Mapping and characterization of an additional NCU04278 allele ......................... 91 17. Loss of NCU04278 upregulates a subset of SET-7 repressed genes near chromosome ends ................................................................................................... 92 18. Genome-wide distribution and level of H3K27 methylation in wild-type, ∆NCU04278, and ∆npf strains ............................................................................... 93 19. The effect of loss of NCU04278 on H3K27 methylation and growth rate ............ 94 20. NCU04278 associates with known N. crassa PRC2 members .............................. 95 21. PAS homologs appear restricted to fungal lineages .............................................. 96 22. Forward genetics identifies a novel gene, epr-1, required for H3K27 methylation-mediated repression ........................................................................... 98 23. Three additional alleles of epr-1 were identified during selection for mutants defective in H3K27 methylation-mediated repression ............................. 99 24. ∆epr-1 and ∆set-7 strains share defects in transcriptional silencing and sexual development ................................................................................................ 100 25. Gene expression and phenotypic analysis of ∆epr-1 and ∆set-7 strains ................ 102 26. epr-1 is not required for H3K27 methylation ........................................................ 103 27. H3K27 methylation levels in ∆epr-1 are comparable to wild type ....................... 104 28. EPR-1 forms telomere-associated foci that are dependent on EED and the BAH domain of EPR-1 .................................................................................... 105 xvii Figure Page 29. EPR-1WT and EPR-1PHD exhibit no difference in distance to telomeres or EPR-1 foci number ................................................................................................ 106 30. EPR-1 directly interacts with H3K27 methylated chromatin through its BAH domain .......................................................................................................... 107 31. EPR-1 associates with H3K27 methylation in vivo and in vitro ............................ 109 32. EPR-1 homologs are present in species beyond fungi ........................................... 110 xviii CHAPTER I INTRODUCTION There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved. - Charles Darwin, On the Origin of Species, 1859 Seen in the light of evolution, biology is, perhaps, intellectually the most satisfying and inspiring science. Without that light it becomes a pile of sundry facts—some of them interesting or curious but making no meaningful picture as a whole. - Theodosius Dobzhansky, The American Biology Teacher, 1973 The ability of an organism to properly regulate its gene expression is critical to its survival. For the single-celled baker’s yeast, Saccharomyces cerevisiae, the functional output of such regulation is limited to concerns of proliferating and mating1. For multicellular organisms, like humans, precise gene regulation also ensures the proper development and maintenance of countless cell types2. If one such human cell should misbehave, e.g. express an improper amount of a gene product, a disease state, like cancer, may result3. Understanding fundamental principles of gene regulation, therefore, may lead to the development of therapies to alleviate conditions associated with aberrant gene expression. 1 The genetic material, DNA, from which genes are transcribed, is located in the nucleus of eukaryotic cells. DNA, along with its associated RNA and protein, constitute a nuclear complex called chromatin. The major constituent of the protein fraction in chromatin consists of histones, of which there are five classes: histone H1, H2A, H2B, H3 and H4. Histones are assembled into nucleosomes that consist of a histone octamer (H2A)2(H2B)2(H3)2(H4)2, which is encircled by ~146 base pairs of DNA. Histones can be post-translationally modified, and it is established that at least some of these modifications have roles in transcriptional regulation4. However, the molecular mechanisms by which these modifications are established, maintained and impact gene expression are largely unresolved. Methylation of histone H3 lysine 27 (H3K27), catalyzed by Polycomb Repressive Complex 2 (PRC2), is known to be associated with maintenance of transcriptional silencing5. Components of PRC2 were originally genetically characterized in the fruit fly, Drosophila melanogaster, as necessary for repression of Hox genes during embryonic development6. H3K27 methylation has been more recently implicated in stem cell identity, cancer, imprinting, and X-chromosome inactivation7. This dissertation investigates the control and function of H3K27 methylation utilizing the filamentous fungus, Neurospora crassa8. N. crassa is an attractive model organism to study H3K27 methylation, in part because more traditional fungal models, such as Schizosaccharomyces pombe and S. cerevisiae, lack this histone mark9. In addition, N. crassa possesses homologues of all core components of mammalian PRC2, and unlike higher eukaryotes these proteins are non-essential for normal development9. 2 The work described in this dissertation contains both published and unpublished co-authored material. Chapter II is an unpublished collaboration with Vincent Bicocca and Eric Selker that examines the role of H3K56 acetylation in ectopic H3K27 methylation. Chapter III was published in volume 7 of the journal eLife in January 2018 with co-authors Kirsty Jamieson, Tereza Ormsby, Neena Leggett, Shinji Honda and Eric Selker. It describes the identification of telomere repeats as a cis-acting signal for H3K27 methylation in N. crassa. Chapter IV is an unpublished collaboration with Elizabeth Wiles and Eric Selker that identifies and characterizes the phenotype of a N. crassa mutant lacking a PRC2 accessory subunit. Chapter V is an unpublished collaboration with Elizabeth Wiles, Saumya De Silva, Gurmeet Kaur, Jeanne Selker, L. Aravind, Catherine Musselman and Eric Selker. It describes the identification and characterization of the first, to our knowledge, ‘reader’ and effector of H3K27 methylation in fungi. 3 CHAPTER II IMPLICATION OF ABERRANT H3K56 ACETYLATION IN ECTOPIC H3K27 METHYLATION This work was a collaboration with Vincent Bicocca. I initiated the investigation, created all relevant Neurospora crassa strains, performed all H3K27me2/3 ChIP-seq experiments, and the summary of results presented was initially drafted by me. Vince Bicocca performed the H3K56ac ChIP-seq experiments and designed the wild-type, codon-optimized histone H3 plasmid. Eric Selker was the principal investigator for this work. Introduction Despite a great deal of effort, a complete understanding of the molecular mechanisms by which PRC2 is recruited to chromatin and subsequently catalyzes H3K27 methylation across the genome in wild-type cells is still unresolved10,11. For this reason, it is of interest to perturb chromatin generally, either genetically or chemically, in an effort to uncover principles that promote or prevent accumulation of H3K27 methylation genome-wide. Our research group previously demonstrated that perturbing constitutive heterochromatin machinery in N. crassa led to redistribution of H3K27 methylation from native sites to constitutive heterochromatin12. This phenomenon appears to be conserved in both plants13,14 and animals15-17. Therefore, determining the features that make 4 constitutive heterochromatin an attractive target for PRC2 in N. crassa may yield insights into PRC2 function in a wide variety of species. Here, in a limited genetic screen, we found that the deletion of neurospora sir two-3 (nst-3) recapitulated the gain of H3K27 methylation at centromeres observed previously in heterochromatin protein one (hpo) mutants12. By introducing an unmodifiable histone H3 (H3K56R) into N. crassa and by removing the sole H3K56 histone acetyltransferase18, we found evidence that this effect is mediated by H3K56 acetylation, in both nst-3 and hpo mutants. Finally, we observed that the gains of H3K27me2/3 at the centromeres were coincident with increases of H3K56 acetylation. Thus, it is possible that H3K56 acetylation plays an important role in the ectopic H3K27 methylation observed in not only N. crassa, but also in plants and animals. Results Deletion of nst-3 results in centromeric H3K27 methylation A previous investigation19 from our research group implicated SIR2 homologues of Neurospora crassa in subtelomeric gene silencing and revealed that H3K27 methylation appeared reduced at some chromosome ends in a mutant defective in three SIR2 homologs. To further investigate the relationship between SIR2 homologues and the control of H3K27 methylation, I performed H3K27me2/3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) on strains bearing deletions of nst-1, nst-3, nst-4, and nst-5 (Fig. 1A). I found that while ∆nst-1, ∆nst-4, and ∆nst-5 strains appeared to have a wild-type-like distribution of H3K27me2/3, ∆nst-3 strains 5 exhibited ectopic gains of H3K27me2/3 at most centromeres, albeit to differing levels, as well as at some regions of interspersed constitutive heterochromatin (Fig. 1A). This phenotype was comparable to, though less pronounced than, that observed in strains defective in constitutive heterochromatin machinery12. Centromeric H3K27 methylation suppressed in ∆nst-3 and ∆hpo strains lacking H3K56 acetylation NST-3 is a homolog of Saccharomyces cerevisiae Hst4p, which acts on acetylated H3K56 (H3K56ac)20. To determine if the K56 residue on histone H3 was necessary for the ectopic H3K27 methylation observed in ∆nst-3 strains, I replaced the endogenous H3 gene with a codon-optimized version of H3 that substituted lysine 56 for arginine (H3K56R). Introduction of H3K56R into ∆nst-3 strains completely suppressed the accumulation of H3K27me2/3 at constitutive heterochromatin (Fig. 1B). It is notable, however, that the H3K56R mutant alone, and in combination with ∆nst-3, did display some ectopic H3K27me2/3, although not at constitutive heterochromatin (Fig. 1B). To further investigate the potential role of H3K56ac in the ectopic H3K27 methylation in ∆nst-3 strains, I created a ∆nst-3 strain bearing an additional deletion of the sole H3K56 acetyltransferase, RTT10918. Similar to ∆nst-3;H3K56R double mutants, ∆nst-3; ∆rtt109 strains did not accumulate H3K27me2/3 at centromeres or interspersed constitutive heterochromatin (Fig. 1B). Taken together, these findings suggest that the aberrant H3K27 methylation observed in ∆nst-3 strains requires H3K56 acetylation. To determine if the previously observed redistribution of H3K27 methylation in strains lacking Heterochromatin Protein 1 (HP1)12 is solely mediated by H3K56ac, I 6 created a ∆hpo;H3K56R double mutant and examined the distribution of H3K27me2/3. While the introduction of H3K56R did partially suppress the redistribution of H3K27me2/3 seen in ∆hpo, it did not fully abolish it (Fig. 1B). This suggests that while H3K56ac may exacerbate the H3K27 methylation phenotype of ∆hpo, other signaling pathways may be involved. H3K56 acetylation appears to accumulate at constitutive heterochromatin in ∆nst-3 strains To determine if there is an association between accumulation of H3K56ac and H3K27me2/3 at constitutive heterochromatin in ∆nst-3 strains, Vince Bicocca performed H3K56ac ChIP-seq on wild-type and ∆nst-3 strains (Fig. 1C). The H3K56ac ChIP-seq showed enrichment at constitutive heterochromatin in the ∆nst-3 strain compared to the wild-type strain, even at regions that do not show ectopic H3K27me2/3. However, H3K56R and ∆nst-3;H3K56R strains subjected to H3K56ac ChIP-seq as negative controls demonstrated that the H3K56ac antibody used is not specific to H3K56ac (Fig. 1C). Therefore, while the most parsimonious explanation is that the ectopic H3K56ac signal detected at constitutive heterochromatin in ∆nst-3 strains is indeed H3K56ac, this is not certain. Furthermore, the fact that H3K56ac appeared to accumulate at all constitutive heterochromatin suggests that while it may be necessary for ectopic H3K27me2/3, it is not sufficient. 7 Discussion The ectopic H3K27 methylation observed at constitutive heterochromatin, especially at centromeres, in ∆nst-3 strains, and subsequent epistasis experiments implicate H3K56ac as a mediator of this phenotype. It remains to be determined if H3K56ac per se is a signal, or it triggers a ‘downstream’ signal for PRC2 activity. Lysine 56 on histone H3 is close to the globular domain of the nucleosome particle, and acetylation of this residue has been implicated in increased nucleosome turnover21-23. Altered nucleosome dynamics at constitutive heterochromatin in ∆nst-3 strains could be responsible for the accumulation of H3K27 methylation. Indeed, nucleosome turnover dynamics can play a critical role in shaping histone methylation patterns genome-wide24. In addition to promoting histone turnover, H3K56ac has been implicated in DNA damage-induced signaling upon replicative stress25. During S-phase, nearly all newly incorporated histones are acetylated at H3K56 in yeast26. Barring DNA damage, this nascent H3K56ac is subsequently removed by histone deacetylases20. Persistently elevated H3K56ac is not only an indicator of DNA damage, but necessary for the DNA damage response in yeast25. However, spontaneous suppressors of a hyperactive DNA damage response in cells with elevated H3K56ac do not necessarily abrogate H3K56ac levels27. Further epistasis experiments, perturbing known components of the DNA damage response pathway in ∆nst-3 strains will elucidate if the accumulation of H3K27 methylation at centromeres is a consequence of the DNA damage response. This is not wild speculation, as Polycomb repressors have known roles in the response to DNA damage28. 8 Materials and Methods Strains, media and growth conditions All N. crassa strains used in this study are listed in Appendix B. Liquid cultures were grown with shaking at 32 °C in Vogel’s minimal medium with 1.5% sucrose29. Crosses were performed at 25 °C on modified Vogel’s with 0.1% sucrose30. Chromatin immunoprecipitation (ChIP) and ChIP-sequencing ChIP and ChIP-sequencing was performed as described in Chapter V Materials and Methods. Active Motif antibodies for H3K27me2/3 (#39536) and H3K56ac (#39281) were used. Replacement of endogenous histone H3 with H3K56R/Q mutants Plasmid 3167, containing a codon-optimized version of N. crassa hH3, was subjected to site-directed mutagenesis using primers 5303 and 5304, or 5305 and 5306 for the introduction of H3K56R or H3K56Q mutations respectively, yielding plasmids 3168 and 3169. The 5’ UTR of hH3 was amplified from genomic DNA with primers 3498 and 5307, the 3’ UTR of hH3 was amplified from genomic DNA with primers 5333 and 5334, and the downstream 3’ flank of hH3 was amplified from genomic DNA with primers 5335 and 5336. The 5’ UTR was PCR stitched to plasmid 3168 or 3169 with primers 3498 and 5337 (Fragment 1). The 3’ UTR was PCR stitched to the nat-1- containing plasmid (3130) with primers 5333 and 4883 (Fragment 2). The 3’ flank was PCR stitched to plasmid 3130 with primers 4884 and 5336 (Fragment 3). Fragment 1 and 9 2 were PCR stitched with primers 3498 and 4883 (Fragment 4). Fragment 3 and 4 were co-transformed into N. crassa strain N2931 and transformants were selected on Nourseothricin-containing media. Bridge to Chapter III The observation that H3K27 methylation accumulates at centromeres in ∆nst-3 strains highlights the fact that PRC2 targeting is not strictly dependent on the underlying DNA sequences. While the AT-rich nature of centromeres may be necessary for this ectopic H3K27 methylation in ∆nst-3 strains, it is clearly not sufficient in wild type. In the next chapter, we further explore the role of underlying DNA sequence and chromosome position on PRC2 activity in wild type strains bearing chromosomal rearrangements. We uncover two fundamentally different classes of H3K27 methylation in N. crassa: telomere-dependent and telomere-independent. 10 CHAPTER III TELOMERE REPEATS INDUCE DOMAINS OF H3K27 METHYLATION IN NEUROSPORA This work was published in volume 7 of the journal eLife in January 2018. I helped target genomic fragments to the csr-1 locus, verified chromosomal translocation breakpoints, created and tested the segmental duplication strain for H3K27 methylation position effects, characterized the tert knock-out strain, targeted telomere repeats to the csr-1 and his-3 locus and examined induced H3K27 methylation, as well as wrote most of the manuscript. Kirsty Jamieson targeted genomic fragments to the his-3 locus and tested for induced H3K27 methylation and characterized H3K27 methylation profile of the chromosomal translocation strains. Tereza Ormsby investigated the gene expression of chromosomal translocation strains. Neena Leggett identified candidate translocation breakpoints and provided bioinformatic support. Shinji Honda created and provided the tert knock-out strain to me. Eric Selker was the principal investigator for this work. Introduction Methylation of lysine 27 on histone H3 (H3K27me) has emerged as an important repressive mark of the Polycomb group (PcG) system, which is critical for development in higher organisms. PcG proteins were initially discovered in Drosophila melanogaster as repressors of homeotic (HOX) genes during early embryogenesis31 and play integral roles in the maintenance of cellular identity and differentiation in a variety of eukaryotes. 11 Moreover, dysfunction of the PcG system commonly leads to disease, including cancer32,33. Biochemical work demonstrated that PcG proteins form two distinct histone- modifying complexes known as Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2)34,35. PRC1 mono-ubiquitinates lysine 119 on histone H2A (H2AK119ub1) with its E3-ubiquitin ligase subunit, Ring1, while PRC2 catalyzes mono-, di-, and trimethylation of histone H3 lysine 27 (H3K27me1/2/3) by its SET-domain component, EZH234,35. PRC2, but not PRC1, is widely conserved in eukaryotes, including the filamentous fungus Neurospora crassa, but is absent in some simple eukaryotes such as the well- studied yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe9. H3K27me2/3 covers approximately 7% of the N. crassa genome, including about 1000 fully-covered genes, all of which are transcriptionally quiescent9,36. The greater than 200 H3K27me2/3 domains, which range from 0.5-107 kb, are widely distributed throughout the genome but are enriched at subtelomeric regions9, as also reported for other fungi37-40. In D. melanogaster, DNA regulatory regions known as Polycomb Response Elements (PREs) recruit PcG proteins to specific chromatin targets to maintain transcriptional silencing41. Recently, similar cis-acting elements have been detected in Arabidopsis thaliana42. In vertebrates and other organisms, however, the mechanism by which PcG proteins are directed to particular loci is elusive43. We show that the control of H3K27 methylation is fundamentally different from that of epigenetic marks in constitutive heterochromatin. Not only is the genomic distribution of H3K27 methylation much more plastic than that of H3K9 methylation12-17,44, but also, unlike DNA methylation and methylation of histone H3K9, which are faithfully methylated de novo when introduced at arbitrary ectopic genomic sites45,46, we show that H3K27 methylation 12 is often position-dependent. We further demonstrate that telomere repeats underpin the observed position effect on H3K27me by showing that loss of telomerase abolishes subtelomeric H3K27me2/3 and that artificial introduction of telomere repeats at interstitial sites trigger deposition of H3K27me2/3. That is, telomere repeats themselves are both necessary for subtelomeric H3K27me2/3 and sufficient to trigger ectopic H3K27 methylation at internal chromosomal sites. Results Analyses of classical chromosome rearrangements suggest that proximity to a chromosome end is key to subtelomeric H3K27 methylation To search for cis-acting sequences that trigger facultative heterochromatin formation in N. crassa, perhaps analogous to PREs in D. melanogaster, we dissected a 47 kb H3K27me2/3 domain on linkage group (LG) VIL. A series of eight, partially overlapping, three kb fragments from this domain were separately targeted to both the histidine 3 (his-3) and the cyclosporin A resistance 1 (csr-1) euchromatic loci in a strain in which we had deleted the endogenous H3K27me2/3 domain (Fig. 3A). H3K27me2/3 chromatin immunoprecipitation (ChIP) followed by qPCR demonstrated the absence of de novo H3K27me2/3 within all eight segments when transplanted to either his-3 or csr-1 (Fig. 3B and C). To address the possibility that the failure to induce H3K27me2/3 in the transplantation experiments was simply due to the size of the test fragments, we utilized strains with large chromosomal rearrangements47. We first examined translocations that 13 involved the H3K27me2/3 domain on LG VIL that we had dissected, starting with UK3- 41, an insertional translocation strain that has an approximately 1.88 Mb segment of LG VR inserted into a distal position on LG VIL (Fig. 2A). The translocation shifted most of LG VIL to a more interior chromosomal position. Interestingly, H3K27me2/3 ChIP-seq of UK3-41 showed an absence of H3K27me2/3 in the region that was displaced from the chromosome end (lost H3K27me2/3; indicated in orange in Fig. 2A). This result is consistent with our finding that transplantation of segments from this region did not induce methylation at his-3 or csr-1 and suggests that the normal methylation of this subtelomeric domain is position-dependent. These results raised the question of whether H3K27me2/3 in this domain absolutely depends on its normal location, or whether the methylation would occur if the region were moved near another chromosome end. To address this possibility, we utilized OY350, a quasiterminal translocation that transfers a distal segment of LG VIL to the end of LG IR (Fig. 2B). Analysis of OY350 by H3K27me2/3 ChIP-seq showed that the translocated LG VIL domain preserved its normal H3K27me2/3 distribution when placed adjacent to the adoptive telomere. This finding supported the hypothesis that H3K27me2/3 in this domain depends on its proximity to the chromosome end and suggested that this requirement is non-specific; the different chromosome end apparently substituted for the native one. A similar situation was observed on LG VIR in OY320 (Fig. 2F). The results described above suggested that proximity to a chromosome end may be sufficient to induce H3K27me. Interestingly, in addition to consistent losses of previously subtelomeric H3K27me2/3 in the examined genomic rearrangements, we 14 identified new H3K27me2/3 domains at novel subtelomeric regions. Striking examples of de novo H3K27me2/3 were independently observed in seven genome rearrangements at their new chromosome ends (Fig. 2B-E and Fig. 4A-C; marked in green). These novel domains of H3K27me2/3 extended an average of approximately 180 kb into the new subtelomeric regions. The only apparent exception concerns translocation UK2-32 (Fig. 4A), which involved the left end of LG V with approximately one Mb of tandemly repeated rDNA that is not shown in the N. crassa genome assembly. The H3K27me2/3 distribution on all chromosomes unrelated to the genome rearrangements was unaltered (Fig. 5). Altogether, our survey of chromosomal translocations strongly suggests that chromosome ends promote the deposition of H3K27me on neighboring chromatin. Identification of position-independent H3K27-methylated domains While domains of H3K27me2/3 are enriched near the ends of chromosomes in N. crassa, substantial domains are also found elsewhere. To examine the possibility of position-independent H3K27me2/3 residing within LG VIR, we examined the distribution of H3K27me2/3 in two translocations that effectively moved LG VIR to the middle of a chromosome. ALS159 is a reciprocal translocation that shifted wild-type LG VIR approximately 4.5 Mb away from the new telomere (Fig. 2C). Although the shifted segment lost the H3K27me2/3 that was previously closest to the chromosome end, the more internal H3K27me2/3 was unaffected by the translocation (Fig. 2C). Similarly, when LG VIR was inserted in the middle of LG III in OY329 (Fig. 2D), most of the H3K27me2/3 on LG VIR was retained. Again, the H3K27me2/3 that was originally 15 closest to the telomere was lost, however, consistent with the idea that subtelomeric H3K27me2/3 domains depend on their proximity to the chromosome ends. To determine if the retention of H3K27me2/3 from LG VIR was unique, we examined the position-dependence of two other H3K27me2/3 domains. In translocation NM149, approximately 600 kb of LG IIL was translocated onto the end of LG VR, effectively shifting the native right arm of LG V about 600 kb away from the chromosome end (Fig. 2E). Like the behavior of LG VIR in ALS159 and OY329, losses of H3K27me2/3 occurred in the most distal section of LG VR, but most H3K27me2/3 was retained, even when moved away from the chromosome end. In addition, a large H3K27me2/3 domain on LG IIIR was shifted approximately 450 kb away from the chromosome end in OY320, yet it did not incur losses (Fig. 2F). Taken together, these findings demonstrate that some H3K27me2/3 domains can be maintained when translocated to internal chromosomal sites, i.e. some H3K27me domains appear to be position-independent. Cis-trans test on H3K27 methylation in a segmental duplication strain reveals position effect The observation that some chromosomal segments that are normally marked with H3K27me2/3 lose the mark when translocated to another genomic location suggested that H3K27me2/3 can be position-dependent. It remained formally possible, however, that the loss of H3K27me2/3 was an indirect effect of the translocation, perhaps due to an altered transcriptional program. To examine the possibility that one or more trans-acting factors could be responsible for the loss of H3K27me2/3 in OY329, we crossed this strain, which 16 is in an Oak Ridge (OR) background, to a highly polymorphic wild-type strain, Mauriceville (MV), to obtain progeny with a segmental duplication, i.e. with the OR translocated LG VIR domain inserted into LG II in a strain that also contained the corresponding region on the native LG VI of MV (Fig. 7A). The high density of single nucleotide polymorphisms (SNPs) in the MV background allowed us to separately map the H3K27me2/3 distribution in the translocated (OR) segment and the wild-type (MV) segment48. SNP-parsed H3K27me2/3 ChIP-seq showed that the duplicated chromosomal segments have distinct H3K27me2/3 profiles (Fig. 7B). The previously subtelomeric H3K27me2/3 that was lost in OY329 did not return in the duplication strain, nor was the loss in this region recapitulated on the native LG VIR of MV. The independent behavior of these homologous segments suggests that the loss of H3K27me2/3 in OY329 was not a result of trans-acting factors, but rather was a result of the translocation itself, i.e. it is a bona fide position effect. Altered gene expression in regions with changed H3K27me2/3 Considering that H3K27me2/3 normally marks transcriptionally quiescent chromatin in N. crassa9, it was of obvious interest to ascertain whether ectopic H3K27me2/3 resulting from chromosomal translocations could cause gene silencing. To determine if the loss or gain of H3K27me2/3 in the translocation strains activated or repressed gene expression, respectively, we performed poly-A mRNA sequencing on wild-type and seven translocation strains (ALS159, AR16, OY329, OY337, OY350, UK2-32, and UK3-41). Fig. 8A and B show gene expression and associated H3K27me2/3 levels in the wild-type and translocation OY350 strains at representative 17 regions where former subtelomeres were moved and new ones created. The gain of H3K27me2/3 on LG VIL in OY350, due to formation of a novel subtelomere, coincided with reduced transcript levels relative to wild-type (Fig. 8A). Conversely, the loss of H3K27me2/3 on LG IR in OY350, due to shifting the subtelomeric region, was associated with an increase in transcript abundance compared to wild-type (Fig. 8B). Results of qPCR analyses of select genes confirmed these findings (Fig. 9). Indeed, a comprehensive analysis of poly-A mRNA-seq data from all seven translocation strains showed increases in gene expression at previously subtelomeric regions that lost H3K27me2/3 and decreases in gene expression at novel subtelomeres that gained H3K27me2/3 (Fig. 8C). These findings demonstrate that chromosomal rearrangements can cause marked changes in both H3K27me2/3 and gene expression of the affected regions. Loss of telomerase abolishes subtelomeric H3K27me2/3 Our analyses of translocation strains demonstrated that domains of H3K27me2/3 can be position-dependent and suggested that a feature of chromosome ends directly or indirectly recruits H3K27me2/3 to subtelomeric regions. To investigate the basis of this, we utilized a mutant lacking the single telomerase reverse transcriptase (tert) that is responsible for all (TTAGGG)n telomere repeats normally found on N. crassa chromosome ends49. Southern analysis of the tert strain using a (TTAGGG)n probe revealed major reductions in the hybridization signals and showed that the majority of telomere fragments present in wild-type were either undetectable or greatly reduced in the mutant (Fig. 10A). In S. pombe, the majority of trt1- (yeast homologue of N. crassa 18 tert) survivors have circularized their chromosomes50. To determine if N. crassa tert survivors also have circular chromosomes, we designed outwardly directed PCR primers to amplify sequences near the ends of each chromosome and tested whether fragments were generated by fusions of the right and left chromosome ends. Indeed, the tert genomic DNA, but not control DNA from a wild-type strain, supported amplification using the divergent primer pairs, confirming intra-chromosomal fusions (Fig. 10B). We performed H3K27me2/3 ChIP-seq to determine whether the subtelomeric domains of H3K27me2/3 were retained or lost in the tert strain and found clear evidence of loss of H3K27 methylation from chromosome ends (Fig. 10C). With the exception of LG V, which is unique in that one of its ends is capped with approximately 150 copies of the approximately 9 kb rDNA repeat51, all chromosomes lost their subtelomeric H3K27me2/3 domains, which typically extend tens of thousands of bp from their ends. Internal H3K27me2/3 domains were not noticeably altered (Fig. 10C and Fig. 11). Sequencing of tert input DNA from the ChIP showed no reduction in coverage near the chromosome ends, confirming that the losses of H3K27me2/3 ChIP signal observed in tert are not caused by chromosome degradation. We conclude that either (TTAGGG)n telomere repeats or linear chromosome ends are required for position-dependent domains of H3K27me2/3 in N. crassa. Internal telomere repeats are sufficient to induce de novo H3K27me2/3 To determine if the presence of telomere repeats is sufficient to trigger the deposition of H3K27 methylation, telomere repeats were separately targeted to the csr-1 and his-3 loci. Due to the repetitive nature of these sequences and our cloning strategy, 19 strains containing a variable number of (TTAGGG) repeats could be obtained through homologous recombination. Sequencing of inserted DNA in eleven independent transformants revealed that the number of inserted telomere repeats ranged from 5 to 23. Native N. crassa telomeres have an average of 20 repeats49. H3K27me2/3 ChIP-qPCR demonstrated that ectopic telomere repeats are sufficient to induce local H3K27me2/3 at the csr-1 and his-3 loci (Fig. 12A and Fig. 13). Even insertion of (TTAGGG)8 triggered some methylation and insertion of (TTAGGG)17 led to a high level of H3K27me2/3 at csr-1. ChIP-seq on the (TTAGGG)17 strain revealed new peaks as far as 170 kb from the insertion site and the semi-continuous H3K27me2/3 domain spanned approximately 225 kb including 30 genes (Fig. 12B and Fig. 14). This is comparable to the size of subtelomeric H3K27me2/3 domains that were lost in the tert strain. Discussion The proper distribution of the facultative heterochromatin mark, H3K27me2/3, deposited by PRC2, is necessary for appropriate gene expression in a variety of plants, animals and fungi11. Unfortunately, the control of H3K27 methylation remains largely unknown. In D. melanogaster, DNA elements known as PREs are important to define domains of H3K27me and associated silencing, but even in this organism, PREs are neither sufficient in all genomic contexts, nor fully penetrant52,53. In addition, it was recently found that deletion of a well-studied PRE in D. melanogaster did not significantly affect either gene silencing or H3K27 methylation at its native locus54. The 20 control of H3K27 methylation is less defined in mammals, in which only a few candidate PRE-like elements have been identified55-57. We took advantage of a relatively simple eukaryote bearing H3K27me, the filamentous fungus N. crassa, to explore the control of this chromatin mark. Conveniently, H3K27me is non-essential in this organism, in spite of being responsible for silencing scores of genes9,58. Unlike the situation with constitutive heterochromatin in N. crassa45,46, we demonstrated that not all facultative heterochromatin is entirely controlled by underlying sequence elements; transplanted gene-sized segments of a H3K27me domain do not, in general, become faithfully H3K27-methylated. However, translocations of large chromosomal segments revealed two distinct classes of transcriptionally repressive H3K27me domains, position-dependent and position- independent. Mechanistic insights from subsequent experiments defined these classes as telomere repeat-dependent and telomere repeat-independent H3K27me. The identification of telomere repeats as potent signals for H3K27 methylation represents a major advance in our understanding of facultative heterochromatin formation in eukaryotes. Our study took advantage of a collection of spontaneous and UV-induced chromosome rearrangement strains of N. crassa that were primarily collected and characterized by David Perkins47. We surveyed representative rearrangement strains in which chromosomal regions containing domains of H3K27me were translocated to novel genomic positions. Strikingly, ChIP-seq revealed multiple cases in which subtelomeric H3K27me2/3 domains completely lost this modification when moved to an interior chromosomal location. Indeed, no exceptions to this rule were found. Generation and 21 analysis of a segmental duplication strain containing differentially marked translocated and normal segments confirmed that the changed distribution of H3K27me2/3 was a bona fide position effect rather than an effect of a trans-acting factor (Figure 2). Moreover, new subtelomeric regions gained H3K27me2/3 over sequences that were previously devoid of this mark (Fig. 2). We conclude that proximity to a chromosome end, per se, somehow induces deposition of H3K27me2/3 in domains that can span hundreds of kilobases. Analyses of polyA+ mRNA from the strains revealed losses of gene expression associated with the new H3K27me2/3 and gains in gene expression in regions that lost this mark; i.e. the changes in H3K27me2/3 were reflected in underlying gene expression levels (Fig. 8). Although gene silencing near chromosome ends, sometimes dubbed “telomere position effect” (TPE) has been observed in fungi19,59-62, D. melanogaster63, Mus musculus64, and human cells65, previous studies did not implicate H3K27me. Interestingly, the extent of heterochromatin spreading from telomeres in N. crassa is substantially greater than previously reported. This could be due to the conspicuous absence of canonical subtelomere sequences in N. crassa49,66-68. Not all organisms that exhibit TPE have H3K27 methylation machinery, but in D. melanogaster, which does, systematic analysis of PcG mutants demonstrated they do not disrupt telomere silencing63. In human cells, TPE is thought to be mediated by histone deacetylation, H3K9 methylation, and heterochromatin protein HP1α67,69. Although the phenomenon of TPE has been studied for decades, our findings may reflect the first documented case of H3K27me-mediated TPE. 22 The ability of chromosome ends to induce large domains of H3K27me2/3 in chromosomal translocation strains motivated us to investigate the role of telomere sequences in the establishment of facultative heterochromatin. We found that deletion of tert, the sole telomerase reverse transcriptase gene, results in a dramatic loss of H3K27me2/3 at subtelomeres, concomitant with loss of (TTAGGG)n repeats and chromosome circularization (Fig. 10). To directly test the possibility that telomere repeats can trigger domains of H3K27me2/3, we inserted an array of telomere repeats at two euchromatic, interstitial sites and used ChIP to check induction of H3K27me2/3. We found that, indeed, even a 48 bp array, (TTAGGG)8, could induce some local H3K27me2/3 at the csr-1 locus. The independent induction of local H3K27me2/3 at the his-3 locus suggests this effect is not position-specific (Fig. 13). Remarkably, a 102 bp array, (TTAGGG)17, induced an H3K27me2/3 domain that covered approximately 225 kb, including 30 genes (Fig. 12). These results strongly suggest that wild-type subtelomeric H3K27me2/3 is dependent on telomere repeats. The recruitment of PRC2 to telomere repeats may be a widespread phenomenon, considering that enrichment of H3K27 methylation at telomeres has been observed in fungi9,37-39, plants70,71 and animals72. Indirect recruitment of PRC2 by telobox-binding transcription factors42 and direct binding of PRC2 to G-quadruplex RNA resulting from transcription of telomere repeats73 represent possible molecular mechanisms. Indeed, it was recently reported that telomeric repeat-containing RNAs (TERRA) directly interact with EZH2 and that H3K27me3 ChIP-seq peaks are strongly correlated with TERRA-associated chromatin in mouse embryonic stem cells74. 23 In addition to telomere repeats capping the ends of chromosomes, telomere repeat-like elements are scattered interstitially in the genomes of many organisms75. In N. crassa, interstitial telomere sequences are rare, limited to less than four tandem repeats, and are not preferentially associated with H3K27me2/3 regions. While S. cerevisiae and S. pombe lack PRC2 components and H3K27me, their internal telomere repeat-like elements can promote heterochromatin formation76,77. The genome of Nicotiana tabacum also has heterochromatic internal telomere repeats, but they lack the H3K27 methylation present at genuine telomeres78. Curiously, insertion of about 130 telomere repeats in Chinese ovary cells failed to significantly alter local transcription79. It should be interesting to study the effects of terminal and interstitial telomere repeats on heterochromatin formation in a variety of organisms. We inferred the existence of position-independent H3K27 methylation from our observation that some H3K27me2/3 domains were unaffected when moved to ectopic chromosomal locations (e.g. see domains on LG IIIR in OY320, LG VR in NM149, and LG VIR in ALS159 and OY329; Fig. 2). The recapitulation of normal H3K27me2/3 profiles at ectopic chromosomal sites is consistent with the possibility that these chromosomal regions contain cis-acting signals that trigger the deposition of H3K27 methylation, perhaps comparable to PREs in D. melanogaster. It will be of interest to define the presumptive elements responsible for such position-independent H3K27me. While there is no consensus sequence for PREs in D. melanogaster, they are composed of unique combinations of binding sites for a variety of factors80. One DNA-binding factor known to affect PcG silencing in D. melanogaster, GRH, has an obvious homolog in N. crassa and could be a candidate for helping establish or maintain position-independent 24 domains of H3K27me2/3 via sequence-specific interactions81-83. It remains possible that deposition of H3K27 methylation at position-independent domains is not directed by sequence-specific elements. In this context, it is worth noting that inhibition of transcription, caused by either exposure to RNA polymerase II inhibitors 84 or deletion of a transcriptional start site85 was found to promote the deposition of H3K27me in mammalian cells. Conceivably, the position-independent domains of H3K27me2/3 in N. crassa could be directed by blocs of inherently low-expressing genes or by some other feature of the region, such as its location in the nucleus, which may be controlled by yet undefined factors. The fact that only about 10% of H3K27me2/3-marked genes are upregulated when the sole H3K27 methyltransferase is removed supports the notion that transcriptional shut-off may precede PRC2 recruitment9,58. Still, low transcriptional activity cannot entirely explain the targeting of PRC2 in N. crassa since many low- expressed genes are not H3K27 methylated. Genome rearrangements are a common occurrence in malignant cells and can drive tumorigenesis through the creation of gene fusions, enhancer hijacking and oncogene amplification86. Our findings suggest that genome rearrangements associated with cancer may additionally impact gene expression through effects on facultative heterochromatin. Indeed, there are already some reports of human diseases being driven by position effects87,88. A great deal of research has focused on how characteristics of local chromatin influence translocation breakpoint frequencies89, but the effects of the resulting translocations on chromatin state are still ill-defined. Altogether, our work shows that changes in genome organization can have sweeping effects on both the 25 distribution of an epigenetic mark and gene expression. Thus, chromosome rearrangements may have unappreciated roles in evolution and cancer etiology. Materials and Methods Strains, media and growth conditions N. crassa strains are listed in Appendix B and were grown and maintained according to standard procedures29. All genome rearrangement strains are available through the Fungal Genetics Stock Center (www.fgsc.net). Deleting and targeting segments of LG VIL A strain containing a 47.4 kb deletion of LG VIL (N4933) was constructed by homologous recombination using primers listed in Appendix C90. For his-3 constructs, segments from LG VIL were PCR-amplified from wild-type (N3752) genomic DNA with primers containing restriction enzyme sites (Appendix C) and directly cloned into pCR (Life Technologies TA Cloning Kit), subcloned into the his-3-targeting vector pBM61 (Appendix D) and transformed into N. crassa strain N573991. For csr-1 replacements, segments from LG VIL were PCR-amplified from wild-type (N3752) genomic DNA with primers containing homology to the 5’ and 3’ flanks of the csr-1 locus (Appendix C). Amplified segments were subsequently assembled by PCR (“PCR-stitched”) to the 5’ and 3’ flanks of the csr-1 locus respectively. Stitched PCR products were co-transformed into N. crassa strain N2931 and transformants were selected on cyclosporin A-containing medium92. 26 Deletion of tert The nat1 gene with the trpC promoter was amplified by PCR using the pAL12- Lifeact as the template with primers 3902 and 1369. The 5’ and 3’ flanking fragments of the tert gene were amplified by PCR with primers 3406-3409, which have specific 29-bp overhang sequence with the 5’ and 3’ nat1 gene with the trpC promoter. The three PCR products were gel-purified, combined and PCR-stitched with primers 3406 and 3409 to construct the knockout cassette. The cassette was gel-purified and transformed into a ∆mus-51 strain (N2929) by electroporation and resulting strains were crossed with a mus- 51+ strain (N3752) to recover progeny with the wild-type allele of mus-51. Targeting telomere repeats to csr-1 and his-3 Concatamers of telomere repeats were amplified in a polymerase chain reaction lacking template as previously described49. Resulting PCR products of ~500 bp were gel- purified and cloned into the pCR4-TOPO TA vector (Life Technologies TOPO TA Cloning kit for Sequencing). Telomere repeats from the cloning vector were PCR- stitched separately to 5’ and 3’ flanks of the targeting locus. For csr-1 targeting, stitched PCR products were co-transformed into strain N5739 and homokaryotic transformants were selected on cyclosporin A-containing medium92. For his-3 targeting, stitched PCR products were co-transformed into strain N2834 and heterokaryotic transformants were selected on medium lacking histidine. 27 ChIP and preparation of libraries ChIP was performed as previously described12. An anti-H3K27me2/3 antibody (Active Motif, 39535) was used for all experiments. ChIPs for Fig. 2-5 and Fig. 8 were performed in biological triplicate. Real-time qPCR was performed as previously described9. ChIP-seq libraries were also prepared as previously described12, except N51, N6089, N6228 and N6383 libraries were subjected to eight cycles of amplification. Sequencing was performed using the Illumina NextSeq 500 using paired-end 100 nucleotide reads for input chromatin and some ChIP experiments. The remainder of the ChIP experiments were sequenced using the Illumina HiSeq 2000 using single-end 100 nucleotide reads or the NextSeq500 using single-end 75 nucleotide reads. All sequences were mapped to the corrected N. crassa OR74A (NC12) genome36 using Bowtie293. ChIP-seq read coverage was averaged over 100 bp sliding (50 bp increment) windows with BEDTools94 and normalized to wild-type by coverage. ChIP-seq data were visualized with Gviz95. To adjust for different sequencing depths between wild-type and translocation samples, a scaling factor was determined for each sample as a ratio of total number of mapped reads in the given sample relative to wild-type. Each scaling factor was then used to normalize ChIP-seq samples. ChIP-seq data were visualized with either IGV96for Fig. 2-5, 7, 10-12 and 14 or Gviz for Fig. 895. Normalized H3K27me2/3 ChIP- seq data were displayed using a 350 or 500 bp sliding widow unless otherwise specified in the figure legend. 28 RNA isolation and preparation of libraries RNA isolation and poly(A)-RNA enrichment, RNA-seq library preparation, and subsequent differential expression analysis were performed with replicate samples as previously described 58. RNA-seq data were visualized with Gviz95. Translocation breakpoint analyses Incongruous-paired and split-read alignments for input DNA samples were found using bwa-mem. The discordant and split-read alignments were analyzed with LUMPY v0.2.9 to determine the location of chromosomal breakpoints97. A list of predicted breakpoints with a minimum call weight of five was generated and calls with an evidence set score <0.05. The chromosomal breakpoints were compared to mapping data determined by recombination mapping47, RFLP coverage and inverse PCR (http://hdl.handle.net/10603/68) to remove calls that were not associated with the translocation of interest. PCR analyses of genome rearrangements ALS159, NM149, OY329, OY320 and UK3-41 were consistent with predicted breakpoint patterns. Southern analysis Southern hybridization analyses were performed as previously described 98. N. crassa telomere restriction fragments were examined as described previously with a HindIII/NotI double digest49. Primers used to make probe are listed in Appendix C. 29 SNP Mapping ChIP-seq data for the duplication strain were converted into fasta format, trimmed to 70 nt, and filtered for 70 nt long reads using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit). Processed data were SNP-parsed with Hashmatch99 using the Mauriceville-Oakridge SNPome (fasta file listing both versions of each SNP)48. The SNPome file was modified to align with read lengths of 70 nt. Only reads with 100% alignment to either genome were kept for further analysis and were allocated into two files according to SNP mapping. Each file was then converted back into fastq format using SeqTK 1.0 (https://github.com/lh3/seqtk) and remapped with Bowtie2 to the genome corresponding with its SNP alignment. To adjust for the variable mapping efficiency of each SNP-parsed file to either Mauriceville or Oakridge, samples were normalized using a scaling factor. The scaling factor was calculated as a ratio of the number of alignments for each SNP-parsed file relative to the file with the smallest number of alignments. H3K27me2/3 profiles were displayed in IGV96 to examine the individual H3K27me2/3 profiles for wild-type and rearranged chromosome segments. Bridge to Chapter IV Our examination of chromosomal rearrangement strains and engineered strains of N. crassa established that at least two classes of H3K27 methylation exist: telomere- dependent and telomere-independent. However, how these two kinds of H3K27 methylation are established and maintained was left unanswered. In the next chapter, using a forward-genetics approach we unexpectedly gain some insight into the apparent 30 division of labor by PRC2 suggested in our findings described in Chapter III. In particular, we identified an accessory subunit of PRC2 that is responsible for subtelomeric H3K27 methylation. 31 CHAPTER IV IDENTIFICATION OF A PRC2 ACCESSORY SUBUNIT REQUIRED FOR SUBTELOMERIC H3K27 METHYLATION IN NEUROSPORA This work was a collaborative effort. I mapped and complemented one of the two identified NCU04278 mutant alleles, analyzed linear growth rates of ∆NCU04278 strains, performed and verified H3K27me2/3 ChIP-seq on ∆NCU04278 strains, verified gene expression changes in ∆NCU04278 strains by RT-qPCR, performed all immunoprecipitations for mass spectrometry, and wrote first draft of this chapter. Elizabeth Wiles created the forward-genetic selection strain and generated the NCU04278 UV alleles, as well as performed the mRNA-seq experiments. Eric Selker was the principal investigator for this work. Introduction Specification and maintenance of cellular fate in higher eukaryotes requires the concerted effort of transcription factor networks and chromatin-modifying complexes. Polycomb repressive complex 2 (PRC2), which catalyzes methylation of lysine 27 on histone H3 (H3K27), is one such chromatin-modifying complex essential for normal development in many organisms100,101. The three core components of PRC2, which are conserved in lineages of plants, animals, and fungi7,102, are EED, SUZ12, and the methyltransferase EZH2. In addition to associating with RBB4/7, human PRC2 interacts with accessory proteins, defining two main subtypes designated PRC2.1 and PRC2.2103. 32 Distinct subcomplexes of PRC2 have also been described in Drosophila melanogaster104- 106, suggesting that this may be a common feature. Here, using a forward genetic selection for factors defective in Polycomb silencing (Chapter V), we isolated two alleles of a gene, NCU04278, necessary for subtelomeric H3K27 methylation in the filamentous fungus, Neurospora crassa. As expected, the loss of H3K27 methylation at the subtelomeres led to concomitant upregulation of subtelomeric genes. Immunoprecipitation followed by mass spectrometry of NCU04278-interacting protein demonstrated that NCU04278 is a PRC2 acccessory subunit, and was therefore deemed PAS. Although PAS homologs appear restricted to the fungal lineage, these findings reiterate the crucial role PRC2 accessory subunits can have in target site selection. Results Isolation, mapping and identification of NCU04278 as potential PcG gene Using a forward genetic selection for factors necessary for Polycomb silencing (Chapter V), we isolated a Hygromycin B- and Nourseothricin-resistant (hnr) mutant (Fig. 15A). We mapped the causative mutation in the hnr strain (Oak Ridge genetic background) by crossing it to the highly polymorphic “Mauriceville” strain107, pooling genomic DNA from progeny with an antibiotic-resistant phenotype, and scoring the percentage of Oak Ridge single nucleotide polymorphisms (SNPs) from whole-genome sequencing data48. We observed an enrichment of Oak Ridge SNPs on the right arm of 33 linkage group (LG) V that included a frameshift mutation (ATG -> TTTG) in NCU04278 (Fig. 15B). To verify that the observed mutation in NCU04278 is the critical mutation in hnr, we targeted a wild-type copy of NCU04278 to the his-3 locus in the hnr mutant background. This ectopic copy of NCU04278 complemented the hnr mutant, i.e., restored Hygromycin B sensitivity (Fig. 15C). In addition, deletion of NCU04278 was sufficient to confer Hygromycin B resistance (Fig. 15C). We subsequently isolated and mapped a second allele of NCU04278 (Fig. 16A), further confirming the involvement of NCU04278 in the antibiotic drug-resistant phenotype. NCU04278 is necessary for silencing subtelomeric H3K27-methylated genes Although our forward genetic selection was designed to identify novel components of the Polycomb repression pathway, mutations in NCU04278 could possibly confer resistance to Hygromycin B and Nourseothricin not through the derepression of the H3K27-methylated antibiotic-resistance genes, but rather through an alternative means, such as more efficient drug efflux108. Alternatively, our selection could have isolated a mutant that had global transcriptional defects, which led to the derepression of hph and nat-1, but was not defective in a specific component of the Polycomb repression mechanism. To determine if loss of NCU04278 has specific defects in Polycomb silencing, we performed mRNA-seq on ∆NCU04278 and wild-type strains in biological replicate and compared the gene expression profiles with previously generated wild-type and ∆set-7 data sets 58. We found that 33 genes were upregulated, and 12 downregulated, greater 34 than two-fold in ∆NCU04278 strains compared to wild-type strains (Fig. 17A,B). Although less than 9% of all genes are H3K27-methylated in a wild-type strain, 64% of the upregulated genes in ∆NCU04278 strains were in this select group. Moreover, there was significant overlap between the upregulated genes in ∆NCU04278 and ∆set-7 strains (P = 6.095 x 10-29), although loss of SET-7 appeared to derepress more H3K27- methylated genes (Fig. 17A), i.e., ∆NCU04278 strains derepress a subset of SET-7 targets. We have previously demonstrated that there are at least two types of H3K27 methylation in N. crassa, telomere-dependent and telomere-independent 109. We reasoned that since loss of NCU04278 only affected a subset of SET-7 targets, NCU04278 may be responsible for specifically silencing either telomere-proximal or telomere-distal H3K27- methylated genes. We therefore analyzed the proximity of H3K27-methylated genes upregulated in ∆NCU04278 and ∆set-7 strains, respectively, and found that the genes derepressed by loss of NCU04278 are closer to the chromosome ends compared to the genes derepressed by loss of SET-7 (Fig. 17C). This suggested that NCU04278 is responsible for silencing telomere-proximal H3K27-methylated genes. We verified the results of our mRNA-seq experiments by performing reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) on RNA isolated from biological triplicates of wild-type, ∆set-7, and ∆NCU04278 strains. In addition to confirming the derepression of the native genes (NCU07152 and NCU05173) replaced by the antibiotic-resistance genes (nat-1 and hph respectively) in the selection strain, we further confirmed a significant increase in gene expression for five out of six H3K27-methylated genes that appeared upregulated by mRNA-seq in both ∆NCU04278 35 and ∆set-7 strains (Fig. 17D). We conclude that NCU04278 represses a subset of SET-7 targets that are telomere-proximal. NCU04278 is necessary for maintenance of subtelomeric H3K27 methylation Considering that the loss of NCU04278 led to the derepression of H3K27- methylated genes near the chromosome ends, we wondered if there was also a concomitant loss of H3K27 methylation at subtelomeric regions. To address this question, we performed H3K27me2/3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) on two ∆NCU04278 siblings and compared it to the wild-type distribution of H3K27me2/3110 (Fig. 18A). We found that the majority of H3K27- methylated genes did not exhibit changes in H3K27me2/3 level in ∆NCU04278 strains compared to wild type (Fig. 18A,B). However, we did detect 284 genes in ∆NCU04278 strains that had at least two-fold reductions in H3K27me2/3 compared to wild-type strains (blue dots; Fig. 18B) and 34 genes that showed at least two-fold greater H3K27me2/3 than wild-type strains (red dots; Fig. 18B). The losses of H3K27me2/3 were concentrated at chromosome ends (Fig. 18A) and were closer to the telomere than the gains (Fig. 19A). To confirm the H3K27me2/3 ChIP-seq results, we performed H3K27me2/3 ChIP followed by qPCR (ChIP-qPCR) on wild-type, ∆set-7, and ∆NCU04278 strains in biological triplicate. We confirmed three regions expected to have no change in H3K27me2/3 (NCU05086, NCU08085, and NCU08251), three regions with expected losses in H3K27me2/3 (NCU05173, NCU07152, and Telomere IIIR), and one region with an expected gain in H3K27me2/3 (NCU07801) (Fig. 18C). Thus NCU04278 is critical for maintaining subtelomeric H3K27 methylation. 36 To determine if there was a correlation between the significant gene expression changes in ∆NCU04278 strains and the losses or gains of H3K27 methylation in ∆NCU04278, we examined the intersection of these two data sets. We found that all but one (NCU08790) of the 21 H3K27-methylated genes upregulated in ∆NCU04278 strains were on the list of genes that lost H3K27 methylation in ∆NCU04278 strains. In contrast, there was no overlap between the genes that gained H3K27 methylation in ∆NCU04278 strains and the genes that were downregulated ∆NCU04278 strains. Analysis of the genes that gained H3K27me2/3 in ∆NCU04278 strains revealed that the majority (88 %) fall into the lowest quartile of gene expression in wild-type strains, suggesting that they were already effectively ‘off’ (Fig. 19B). In summary, the losses of H3K27 methylation in ∆NCU04278 strains are associated with, but not sufficient for, increased gene transcription, and the gains of H3K27 methylation in ∆NCU04278 strains are mostly on genes that are normally poorly transcribed. NCU04278 is an accessory subunit of PRC2 To determine if NCU04278 acts in a complex, we immunopurified NCU04278- 3xFLAG from N. crassa lysate and identified its copurifying proteins by mass spectrometry. We identified NCU04278 itself, as well as the four known components of PRC2: SET-7, SUZ12, EED, and NPF (Fig. 20A)9. We also immunopurified SUZ12- 3xFLAG and a negative control (3xFLAG-control), and analyzed their copurifying proteins by mass spectrometry. In addition, we examined previously collected mass spectrometry data from a 3xFLAG-EED purification 9. The purifications of the known PRC2 components, SUZ12 and EED, yielded NCU04278 as well as the rest of the known 37 members of the complex (Fig. 20A). The 3xFLAG-control purification did identify peptides from NPF, but did not detect any peptides from SET-7, SUZ12, EED, or NCU04278 (Fig. 20A). Besides the proteins listed in Fig. 20A, the only other proteins with peptides detected in all the NCU04278, SUZ12, and EED purifications, but absent in the control. were NCU01249 (importin α), NCU02407 (dihydrolipoyl dehydrogenase), and NCU06482 (pyruvate dehydrogenase). Thus, it appears that PRC2 in N. crassa can form a five-member complex and that NCU04278 is an accessory component. For this reason, we have deemed NCU04278 as PAS (PRC2 accessory subunit) (Fig. 20B). Discussion Ever since the identification of Polycomb response elements (PREs) in Drosophila melanogaster, sequences that reliably induce H3K27 methylation41, there has been an intense search for other cis- and trans-acting factors that direct PRC2 activity in a wide variety of organisms. In plants, this line of inquiry has been fruitful, with researchers demonstrating the direct interaction of PRC2 with transcription factors that bind specific DNA sequences42,111,112. In mammals, unmethylated CpG islands can apparently recruit PRC2113-115, but the mechanism of this recruitment is unclear. Non- core subunits of mammalian PRC2.1 and PRC2.2 are collectively required to target PRC2116,117, but how these structurally distinct components can independently target the same genomic sites is unsettled. Defining the full complement of cis- and trans-acting factors and how they specifically recruit PRC2 remains an open challenge. 38 Utilizing a relatively simple eukaryote, the fungus N. crassa, we performed a forward genetic selection to identify novel factors implicated in Polycomb repression. This yielded an unknown, fungal-specific, accessory component of PRC2, PAS (NCU04278), that is necessary for subtelomeric H3K27 methylation and associated gene silencing. Interestingly, we had already observed an H3K27 methylation defect similar to that observed in ∆pas strains in strains lacking the PRC2 component, NPF9. Indeed, H3K27me2/3 ChIP-seq profiles from ∆pas and ∆npf strains bear a striking resemblance (Fig. 18A), although they are clearly different. It was not certain that the lack of NPF in PRC2 was the direct cause of the observed defects in ∆npf strains, as this protein also associates with other complexes38, ∆npf strains exhibit greater changes in gene expression than ∆set-7 strains58 and ∆npf strains display growth defects not seen in other PRC2 member knockout strains9. It is notable that ∆pas strains, although quite similar to ∆npf strains in H3K27 methylation defects, have more limited changes in gene expression (Fig. 17A,B) and no detectable growth defect (Fig. 19C). Despite difficulties in establishing direct or indirect effects of NPF loss on PRC2 recruitment in vivo in N. crassa, such experiments are not feasible in mammalian cell lines as RBBP4/7 are essential for proliferation10. Our research group has previously identified telomere repeats, (TTAGGG)n, as effective inducers of H3K27 methylation at chromosome ends in N. crassa109. This subtelomeric H3K27 methylation is largely lost in ∆pas, ∆npf and ∆tert (telomerase) strains (Fig. 18A)109. Although the exact link between the cis-acting telomere repeats and induced H3K27 methylation remains unclear, the identification of PAS should help bridge this gap in our knowledge. 39 Materials and Methods Strains, media and growth conditions All N. crassa strains used in this study are listed in Appendix B. Preparation of media and growth conditions for experiments were carried out as previously described (Chapter V). Selection for mutants defective in H3K27 methylation-mediated silencing Conidia from strain N6279 were mutagenized with ultraviolet light and challenged with Hygromycin B and Nourseothricin to select for drug-resistant colonies as previously described (Chapter V). Primary mutants were rendered homokaryotic by crossing to strain N3756. Whole genome sequencing, mapping and identification of NCU04278 alleles This was performed as previously described (Chapter V). Briefly, drug-resistant, homokaryotic mutants were crossed to a “Mauriceville” strain107 and drug-resistant progeny were pooled for whole genome sequencing (SRA accession: PRJNA559544). Mapping of the causative mutations was performed as previously described48. FreeBayes and VCFtools were used to identify novel genetic variants present in our pooled mutant genomic DNA118,119. 40 Chromatin immunoprecipitation (ChIP), ChIP-qPCR, and ChIP-seq H3K27me2/3 ChIP using anti-H3K27me2/3 antibody (Active Motif, 39536) was performed as previously described (Chapter V) and the isolated DNA used for qPCR or prepared for sequencing (Chapter V). Mapping, visualization, and analysis of H3K27me2/3 ChIP-sequencing reads was performed as previously described (Chapter V). RNA isolation, RT-qPCR, and mRNA-seq Extraction of total RNA from germinated conidia was performed as previously described (Chapter V) and either used for cDNA synthesis and subsequent qPCR (Chapter V) or mRNA-seq library preparation58. Mapping and analysis of gene expression levels was performed as previously described (Chapter V). Immunoprecipitation followed by mass spectrometry (IP-MS) Approximately 1x109 conidia collected from 7-10 days of growth of strain N7807 (over-expressing NCU04278::C-Gly::3xFLAG) or strain N4666 (endogenous suz12::C- Gly::3xFLAG) in a 250 mL flask containing 50 mL of Vogel’s minimal medium containing 1.5% sucrose and 1.5% agar was filtered through sterile cheesecloth. Filtered conidia were used to inoculate 1 L of Vogel’s minimal medium containing 1.5% sucrose and then grown for 16 hours shaking (150 RPM) at 30 °C. Tissue was collected by filtration using a Buchner funnel and washed with water. Dry tissue (~10 g) was ground using a 6870 Freezer/Mill Cryogenic Grinder (SPEXSamplePrep). Ground tissue was added to 40 mL Extraction Buffer (EB) (50 mM HEPES [pH=7.5], 150 mM NaCl, 10 41 mM EDTA, 10% glycerol, 0.02% NP-40, 1X Halt Protease Inhibitor Cocktail (Thermo Scientific, 78438)) to achieve a final volume of 50 mL, and then suspended by rotation at 4 °C for 1.5 hours. Insoluble material was pelleted with one 10-minute centrifugation step at 2,000 RPM and two consecutive 10-minute centrifugation steps at 8,000 RPM. Soluble material was pre-cleared with 250 µL of Protein A agarose (Invitrogen, 15918014) equilibrated in EB with rotation for 1 hour at 4 °C. The Protein A agarose was pelleted by centrifugation at 2,000 RPM and the supernatant collected, which was then incubated with 400 µL of ANTI-FLAG M2 affinity gel (Sigma-Aldrich, A2220) equilibrated with EB overnight, rotating at 4 °C. Resin was pelleted by centrifugation at 1,000 RPM and supernatant removed. Resin was washed with EB, rotated at 4 °C for 10 minutes, and pelleted with a 1,000 RPM spin five consecutive times. All liquid was removed after the final spin. Protein was eluted twice from the resin by incubating with 300 µL of 500 µg/mL 3X Flag Peptide (APExBIO, A6001) rotating at 4 °C for 20 minutes, and one final wash with 300 µL of EB. Eluate was precipitated with trichloroacetic acid (10% final concentration) on ice for 1 hour, pelleted by centrifugation at 14,000 RPM, and washed three times with ice-cold acetone. Pellet was air-dried by placing in heat block at 100 °C for 30 seconds. Samples were sent to and processed by the UC Davis Proteomics Core Facility for mass spectrometry and subsequent analysis. Replacement of NCU04278 with trpC::nat-1 The 5’ and 3’ flanks of NCU04278 were PCR-amplified from wild-type genomic DNA with primer pairs 6405 and 6406 (5’) and 6409 and 6410 (3’). The 5’ and 3’ flanks were separately PCR-stitched to plasmid 3237 (source of trpC::nat-1) using primer pairs 42 6401 and 4883, and 4882 and 6351, respectively. These two ‘split-marker’ PCR products were co-transformed into strain N2930 and NCU04278 replacements were selected on Nourseothricin-containing medium. Generation of a N. crassa strain expressing NCU04278::3xFLAG NCU04278 was PCR-amplified from wild-type genomic DNA with primers 6397 and 6398, and cloned into plasmid 2401 120 using XbaI and PacI restriction sites to create plasmid 3340. Plasmid 3340 was subjected to Sanger sequencing using primers 6397, 6407, 6426, 6427, and 6715 to verify that the sequence matched that of wild-type. Plasmid 3340 was linearized with NdeI and targeted to his-3 in N6762, as previously described 91. A his-3+ primary transformant was then crossed to N7742 to generate N7807. Bridge to Chapter V In this chapter, we utilized a powerful forward genetic selection, developed by Elizabeth Wiles, and identified an unknown accessory subunit of PRC2 that is necessary for subtelomeric H3K27 methylation. H3K27-methylated genes near the chromosome ends lose their methylation and a subset of these are derepressed. While this result does gives us some insight into the workings of PRC2 targeting/activity, the mechanism of how H3K27 methylation mediates repression is unresolved. In the next chapter, we address this issue when we identify a ‘reader’ and effector of H3K27 methylation using 43 the aforementioned genetic selection. This protein, which we deemed EPR-1, is not limited to fungi but exists in a variety of eukaryotic lineages. 44 CHAPTER V EVOLUTIONARILY ANCIENT BAH-PHD PROTEIN MEDIATES POLYCOMB SILENCING This work was a collaborative effort. I mapped and complemented one of the four identified epr-1 mutant alleles, analyzed linear growth and sexual development phenotypes of ∆epr-1 strains, performed H3K27me3 westerns and H3K27me2/3 ChIP- seq on ∆epr-1 strains, created GFP-EPR-1-expressing strains and performed some microscopy, created EPR-1-Dam-expressing strains and performed DamID-seq and Southerns, as well as wrote initial draft of the main body of the manuscript. Elizabeth Wiles created the forward-genetic selection strain and generated the epr-1 UV alleles, verified the mRNA-seq results, verified the H3K27me2/3 ChIP-seq results, performed and verified GFP-EPR-1 ChIP-seq results, cloned BAH domain of EPR-1, and contributed significantly to manuscript. Saumya De Silva, under supervision of Catherine Musselman, performed NMR titration experiments. Gurmeet Kaur, under supervision of L. Aravind, performed the phylogenetic analysis. Jeanne Selker performed some microscopy experiments. Eric Selker was the principal investigator for this work. Introduction The establishment and maintenance of transcriptionally repressive chromatin is critical for the development of multicellular organisms121-124. Polycomb group (PcG) proteins, originally discovered in Drosophila melanogaster31, form multiple complexes 45 that maintain such chromatin repression125. Although the composition of PcG complexes varies, a few core constituents define two major classes of chromatin-modifying complexes, namely Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2)7. According to the ‘classical model,’ PcG-mediated gene silencing is initiated by targeting of PRC2 to chromatin80, which catalyzes methylation of H3K2734. Canonical PRC1, which contains a chromodomain protein (e.g. Polycomb in Drosophila melanogaster and CBX2/4/6-8 in mammals), recognizes tri-methylated H3K27126, catalyzes monoubiquitination of neighboring histone H2A lysine 119 by RING1A/B35, and promotes chromatin compaction127,128. In reality, this hierarchical recruitment model is an oversimplification, as PRC1 can be recruited to PcG targets irrespective of PRC2 activity129 and PRC1 presence is required for stable PRC2 association at many Polycomb Response Elements in Drosophila melanogaster130. Interdependence of these complexes has limited our understanding of their respective roles and the function of their associated chromatin ‘marks’ on gene repression. While plants and animals utilize distinct sets of accessory proteins to recognize methylated H3K27126,131-133, they are generally thought to mediate repression in the context of a canonical PRC1 complex7,134-136. In fungal lineages that employ H3K27 methylation as a repressive chromatin mark, however, core PRC1 components are notably absent7. This raises the question of how H3K27 methylation mediates repression in the absence of PRC1. It suggests that either: 1. H3K27 methylation per se may be repressive, or 2. There is a ‘reader’ of H3K27 methylation that functions outside the context of canonical PRC1. 46 To elucidate the repressive mechanism of H3K27 methylation in fungi, we developed and employed a forward genetics approach to identify effectors of Polycomb repression using Neurospora crassa. H3K27 methylation covers approximately 7% of the N. crassa genome and is responsible for the repression of scores of genes9,58. We found four mutant alleles of an undescribed gene (NCU07505) that we show is critical for H3K27 methylation-mediated silencing and therefore named it effector of Polycomb repression 1 (epr-1). It encodes a protein with a bromo-adjacent homology (BAH) domain and plant homeodomain (PHD) finger. Although epr-1 mutants display phenotypic and gene expression changes similar to strains lacking PRC2 components, H3K27 methylation is essentially unaffected. We demonstrate that EPR-1 forms nuclear foci, reminiscent of Polycomb bodies137, and its genomic distribution is limited to, and dependent upon, H3K27-methylated chromatin, which it recognizes directly through its BAH domain. Finally, we discover that EPR-1 orthologs are widely distributed across eukaryotes, contrary to previous reports136,138,139, suggesting an ancient role of EPR-1 homologs in Polycomb repression that was then lost on multiple occasions in certain lineages. Results Genetic selection for factors necessary for H3K27 methylation-mediated repression In an effort to identify factors required for H3K27 methylation-mediated repression, we engineered a strain of N. crassa in which we replaced the open reading frames of two PRC2-repressed genes58, NCU05173 and NCU07152, with the antibiotic- 47 resistance genes hph and nat-1, respectively (Fig. 22A). Strains that bear these gene replacements and lack the H3K27 methyltransferase (SET-7) are resistant to Hygromycin B and Nourseothricin, whereas a wild-type strain with these gene replacements is sensitive to these drugs (Fig. 22C). We subjected conidia collected from such an antibiotic-sensitive, otherwise wild-type strain to ultraviolet (UV) mutagenesis and selected for mutants that derepressed both the hph and nat-1 genes (Fig. 22B). One mutant isolated in this manner and characterized here is effector of polycomb repression 1 (epr-1) (Fig. 22C). Mapping and identification of epr-1 as NCU07505 In order to map and identify the causative mutation in the epr-1UV1 mutant, we crossed epr-1UV1, which is in an Oak Ridge genetic background, to a highly polymorphic wild-type strain named “Mauriceville”107. We then pooled the genomic DNA from Hygromycin B-resistant progeny and subjected it to whole-genome sequencing (~15x coverage; Fig. 22D). When we scored the percentage of Oak Ridge single nucleotide polymorphisms (SNPs) across the genome48, we found a region on linkage group (LG) I that was enriched for Oak Ridge SNPs and included an early stop mutation (Q206*, CAG->TAG) in NCU07505 (Fig. 22E). To verify that the early stop in NCU07505 is the causative mutation in epr-1UV1, we targeted a wild-type copy of NCU07505 to the his-3 locus in the epr-1UV1 mutant background. This ectopic copy of NCU07505 complemented the mutation, i.e., it restored drug sensitivity (Fig. 22F). In addition, we found that deletion of NCU07505 resulted in resistance to Hygromycin B, similar to the epr-1UV1 strain (Fig. 22F). We subsequently 48 isolated and characterized three additional alleles of epr-1 generated in the mutagenesis, further supporting the notion that mutations in NCU07505 support drug resistance (Fig. 23). EPR-1 and SET-7 repress an overlapping set of H3K27-methylated genes Although our selection was designed to isolate mutants with specific defects in Polycomb repression, we could conceivably recover mutants that globally altered transcription or led to antibiotic resistance independent of hph or nat-1 upregulation. To determine if EPR-1 was specifically required for repression of H3K27-methylated genes, we performed mRNA-seq on ∆epr-1 siblings and compared the gene expression profile to previously published wild-type and ∆set-7 data sets58. We found that 632 genes were upregulated and 974 genes were downregulated greater than two-fold in ∆epr-1 strains compared to wild-type strains (P < 0.05) (Fig. 25A). The upregulated gene set in ∆epr-1 was significantly enriched for H3K27-methylated genes (X2(1, N = 632) = 40.8, P = 1.684 x 10-10)(Fig. 25A), and H3K27-methylated genes upregulated in both ∆epr-1 and ∆set-7 significantly overlapped (P = 1.436 x 10-16) (Fig. 24A). To verify our mRNA-seq results, we performed reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) on RNA isolated from biological triplicates of wild-type, ∆set-7, and ∆epr-1 strains. Five of six examined H3K27-methylated genes found upregulated in both ∆set-7 and ∆epr-1 strains by mRNA-seq were confirmed by RT-qPCR (Fig. 24B and Fig. 25B). In contrast, only one out of six H3K27-methylated genes found exclusively upregulated in ∆set-7 or ∆epr-1 by mRNA-seq was confirmed by RT-qPCR (Fig. 25C-D). Thus, these 49 data show that loss of EPR-1 derepresses a significant number of H3K27-methylated genes that are also upregulated in strains lacking H3K27 methylation. ∆epr-1 and ∆set-7 strains share a defect in sexual development Since ∆epr-1 and ∆set-7 strains exhibit similar transcriptional profiles, we wondered if they also shared vegetative growth and sexual development phenotypes as well. To assess if ∆epr-1 strains have an altered vegetative growth rate, we measured linear growth rates of wild-type, ∆set-7 and ∆epr-1 strains with ‘race tubes’140. We confirmed that ∆set-7 strains do not have a linear growth defect9 and found that ∆epr-1 strains also grow at wild-type rates (Fig. 25E). Loss of SET-7 has been implicated in promoting sexual development in mutants that are homozygous sterile141. To determine if ∆set-7 and/or ∆epr-1 strains aberrantly promote sexual differentiation in the absence of a mating partner, we singly inoculated crossing plates30 with wild-type, ∆set-7 or ∆epr-1 strains. After two weeks of unfertilized growth at 25 °C, we observed the development of few false perithecia with wild-type controls, whereas ∆epr-1 and ∆set-7 developed approximately 10- and 100-fold more false perithecia than wild type, respectively (Fig. 24C and Fig. 25F). These data suggest that EPR-1, and SET-7 to a greater extent, repress premature sexual development, which is reminiscent of fertilization-independent seed development observed in plant Polycomb mutants142,143. H3K27 methylation is essentially normal in ∆epr-1 As a first step to assess if the transcriptional silencing and sexual development defects shared between ∆epr-1 and ∆set-7 strains were due to a common global loss of 50 H3K27 methylation, we performed a western blot on whole cell lysates to detect H3K27me3 in wild-type, ∆set-7, and ∆epr-1 strains. The total levels of H3K27me3 in ∆epr-1 strains were comparable to that in wild type (Fig. 26A and Fig. 27A-C). However, because only a subset of H3K27-methylated genes are derepressed in both ∆epr-1 and ∆set-7 strains, we wanted to know if H3K27 methylation might be specifically lost at upregulated genes in ∆epr-1 strains. To examine this possibility, we performed H3K27me2/3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) on two ∆epr-1 siblings and compared the data to that for wild type110. We found that the global distribution of H3K27me2/3 in ∆epr-1 appeared to mirror that of wild type (Fig. 26B). Comparison of H3K27me2/3 levels associated with individual genes showed good agreement between the averaged wild-type and ∆epr-1 data sets (R2 = 0.9105), and within replicate data for wild-type (R2 = 0.9272) and ∆epr-1 (R2 = 0.8941) strains (Fig. 26C and Fig. 27D-E). We did, however, identify 29 genes with a greater than two-fold decrease and nine genes with a greater than two-fold increase in H3K27me2/3 levels in ∆epr-1 compared to wild type. Interestingly, none of these 38 genes with altered H3K27 methylation were classified among the upregulated or downregulated gene sets in the mRNA-seq analysis of ∆epr-1. To validate the H3K27me2/3 ChIP-seq results, we performed H3K27me2/3 ChIP followed by quantitative Polymerase Chain Reaction (ChIP-qPCR) on wild-type, ∆set-7, and ∆epr-1 strains in biological triplicate. These data confirmed wild-type levels of H3K27me2/3 at a subtelomere (Tel IL) and at the genes replaced by the antibiotic-resistance markers (NCU07152 and NCU05173), and also corroborated the loss (NCU08834) and gain (NCU02856) of H3K27me2/3 observed in the ChIP-seq of ∆epr-1 strains (Fig. 26D). Altogether, these data show that the 51 derepression of H3K27-methylated genes in ∆epr-1 strains is not due to concomitant loss of H3K27 methylation. EPR-1 forms telomere-associated foci dependent on EED To localize EPR-1 in vivo, we used the ccg-1 promoter to drive expression of wild-type EPR-1 fused with GFP at its N-terminus (EPR-1WT)120 in a strain that had fluorescent markers for the nuclear membrane (ISH1), telomeres (TRF1), and centromeres (CenH3)58. We found that EPR-1WT was restricted to the nucleus and formed distinct foci that were typically closely associated with TRF1 foci (Fig. 28A). EPR-1WT foci were significantly closer to telomeres as compared to centromeres (negative control; P = 0.0403) (Fig. 29A), and the number of EPR-1WT and TRF1 foci per nucleus were not statistically different (P = 0.7422), although the majority of nuclei examined had more EPR-1WT than TRF1 foci (Fig. 29B-C). Considering that H3K27 methylation is predominantly present near chromosome ends in N. crassa9, it is not unexpected that a putative PcG protein, such as EPR-1, would co-localize with the telomere marker, TRF1. To determine if the formation of EPR-1WT foci was dependent on H3K27 methylation, we introduced a deletion of eed, a gene encoding a component of PRC2 necessary for catalytic activity9. Strains bearing this deletion lacked distinct nuclear foci of EPR-1WT and instead displayed a diffuse nuclear distribution of EPR-1WT (Fig. 28B). Thus, an intact PRC2 complex or H3K27 methylation is required for proper EPR-1WT subnuclear localization. 52 An intact BAH domain is required for normal nuclear distribution of EPR-1 EPR-1 is predicted144 to have a BAH domain and PHD finger, protein modules implicated in chromatin engagement145,146. To determine if these domains are necessary for the formation of the EPR-1WT foci, we created GFP-EPR-1 constructs in which a previously identified critical tryptophan in either the BAH domain (EPR-1BAH) or PHD finger (EPR-1PHD) was replaced with an alanine147,148. We found that EPR-1BAH displayed the same diffuse nuclear distribution as EPR-1WT in a ∆eed background, consistent with the possibility that the BAH domain mediates interaction with H3K27-methylated chromatin (Fig. 28C). In contrast, EPR-1PHD still formed nuclear foci that were equivalent to EPR-1WT in number and proximity to TRF1 foci (Fig. 28D and Fig. 29D-E), demonstrating the nonessential nature of this conserved tryptophan residue for the normal nuclear distribution of EPR-1. EPR-1 localizes to H3K27 methylation genome-wide Since proper subnuclear localization of EPR-1WT appeared to require H3K27 methylation, we performed ChIP-seq to determine if EPR-1WT genomic targets coincided with H3K27me2/3 throughout the genome (Fig. 30A). Results of EPR-1WT ChIP-seq appeared to match the distribution of H3K27me2/3 in wild type (Fig. 30A) and we found good correlation between EPR-1WT and H3K27me2/3 relative sequencing coverage over each gene (R2 = 0.8675) (Fig. 31A). We also examined the genomic distribution of EPR- 1PHD and EPR-1BAH mutant alleles (Fig. 30A), which were expressed at comparable levels (Fig. 31B-C). The ChIP-seq coverage of EPR-1PHD was still enriched at H3K27- methylated genes, albeit less so than EPR-1WT, while the EPR-1BAH ChIP-seq did not 53 show enrichment (Fig. 31D). To validate the ChIP-seq of GFP-EPR-1-expressing strains, we performed ChIP-qPCR for representative regions (Fig. 30B). Consistent with the ChIP-seq data, EPR-1WT, and EPR-1PHD to a lesser degree, were enriched at examined regions bearing H3K27 methylation. In addition, the ChIP-qPCR confirmed that EPR- 1BAH, as well as EPR-1WT in a ∆eed background, lack such enrichment. As an orthogonal approach to ChIP, we determined the chromatin targets of EPR- 1 by fusing an E. coli DNA adenine methyltransferase (Dam)149 to the C-terminus of endogenous EPR-1 and assayed adenine-methylated DNA fragments by sequencing (DamID-seq)150 (Fig. 30A). Using DamID-seq of EPR-1WT-Dam and methyl-sensitive restriction enzyme Southern blots, we found that EPR-1WT-Dam localizes to H3K27- methylated genes and this is dependent upon EED (Fig. 30A and Fig. 31E-F). Mutation of the PHD finger in the EPR-1-Dam fusion did not abolish its targeting to H3K27- methylated chromatin (Fig. 30A). These results were consistent with our ChIP-seq findings. We conclude that EPR-1 localizes to H3K27-methylated regions of the genome and that proper recruitment of EPR-1 to chromatin requires both an intact BAH domain and the integral PRC2 component, EED. Both the BAH domain and PHD finger of EPR-1 are necessary for gene repression Our localization studies of EPR-1PHD and EPR-1BAH, while suggestive, did not directly test the role of the PHD finger and BAH domain of EPR-1 in H3K27 methylation-mediated silencing. We therefore utilized our antibiotic-resistance reporters, used in the initial selection, to test more directly their possible involvement in gene repression. We targeted ectopic copies of epr-1WT, epr-1BAH or epr-1PHD to the his-3 locus 54 in an epr-1UV1 strain bearing the antibiotic-resistance genes and scored drug resistance. Whereas epr-1WT restored sensitivity to Hygromycin B and Nourseothricin, epr-1BAH remained resistant to both drugs (Fig. 30C). In contrast, introduction of epr-1PHD apparently re-silenced the hph, but not the nat-1, antibiotic-resistance gene (Fig. 30C). This suggests that while the PHD finger of EPR-1 is not essential for recruitment to H3K27 methylated chromatin, it is not entirely dispensable for gene silencing. BAH domain of EPR-1 binds to H3K27me3 in vitro To test, directly, whether the BAH domain of EPR-1 recognizes H3K27 methylation, we cloned, expressed, and purified a HIS-SUMO-BAHEPR-1 fusion protein for use in nuclear magnetic resonance (NMR) spectroscopy experiments. We found that the BAHEPR-1 domain alone was unstable once cleaved from the HIS-SUMO tag and therefore we used the HIS-SUMO-tagged BAHEPR-1 in all subsequent experiments. An initial 1H–15N-heteronuclear single quantum coherence (1H–15N HSQC) spectrum revealed that the fusion protein was well folded (Fig. 31H). An overlay with an 1H–15N HSQC spectrum of 15N labelled HIS-SUMO tag alone was used to determine which peaks belonged to BAHEPR-1 (Fig. 31H). Addition of increasing concentrations of an H3K27me3 peptide to the HIS-SUMO-BAHEPR-1 fusion protein led to significant chemical shift perturbations (CSPs), indicating binding (Fig. 30D-E). Importantly, the perturbed resonances belonged exclusively to BAHEPR-1. We conclude that the BAH domain of EPR-1 associates with an H3K27me3 peptide. Due to protein stability problems, we were unable to calculate an accurate dissociation constant (Kd); however, the CSPs appeared consistent with a high micromolar Kd. 55 EPR-1 is a homolog of plant EBS/SHL and widely distributed across eukaryotes To determine if EPR-1 orthologs exist outside of N. crassa, we performed sequence similarity searches to identify homologs, followed by phylogenetic and domain architectural analysis of those to identify genuine orthologs. Consequently, we were able to identify orthologs in various fungal species as well as a wide range of other eukaryotes (Fig. 32A). Notably, we determined the Arabidopsis thaliana paralogs EBS and SHL as orthologs of EPR-1 in our analyses, which have been erroneously reported as plant- unique proteins136,138,139. Similar to EPR-1, the plant paralogs, EBS and SHL, bind H3K27 methylation and have been implicated in gene repression132,133,136. As one approach to investigate if other EPR-1 orthologs may have roles independent of H3K27 methylation, we checked if any species has an EPR-1 ortholog but lacks a SET-7 (H3K27 methyltransferase) ortholog. With the exception of Chytridiomycota and Fonticula lineages, in which the presence of SET-7 homologs was deemed ambiguous due to lack of a definitive pre-SET domain, all examined species with EPR-1 homologs had clear SET-7 homologs (Fig. 32A). This result is consistent with EPR-1 orthologs mediating H3K27 methylation-based repression in a wide variety of species. In contrast to EPR-1, A. thaliana EBS is not entirely restricted to H3K27- methylated genes. Indeed, the majority of EBS-bound genes are devoid of H3K27me3 and instead are associated with an ‘active’ chromatin mark, H3K4me3151, via the PHD finger of EBS132. To understand this discrepancy, we examined the underlying protein sequence of the PHD finger of EPR-1. Whereas aromatic residues implicated in methylated histone recognition in the BAH domain152 are present in both EPR-1 and the 56 plant paralogs, comparable residues in the PHD finger133,153,154 are present in EBS and SHL, but lacking in EPR-1 (Fig. 32B, highlighted in red). Furthermore, a single amino acid substitution replacing an aromatic tyrosine residue with an alanine residue in the PHD finger of a plant SHL was sufficient to diminish in vitro H3K4me3-binding greater than two-fold133. Intriguingly, this residue corresponds to the naturally occurring alanine 275 in EPR-1 (Fig. 32B). This suggests that while many species have EPR-1 homologs, they may not necessarily be bivalent histone readers like plant EBS and SHL. Discussion Deciphering the basic mechanism(s) of Polycomb repression has been difficult, in part, due to the diversity103, redundancy155, as well as interdependence130 of protein players involved. For this reason, the model organism N. crassa, which employs H3K27 methylation catalyzed by PRC2 for gene repression9, yet conspicuously lacks PRC1 components7, represents an ideal organism to uncover fundamental aspects of Polycomb silencing. Here we have identified, to the best of our knowledge, the first known reader and effector of H3K27 methylation in fungi, EPR-1. This provides insight into how Polycomb silencing can function in the absence of PRC1. Our ChIP and DamID results demonstrated that EPR-1 co-localizes with H3K27 methylation genome-wide and cytological examination revealed that GFP-EPR-1 forms approximately 3-5 foci per nucleus. This implies that domains of H3K27 methylation within and between the seven N. crassa chromosomes generally self-associate. This is consistent with the observed intra- and inter-chromosomal contacts among H3K27- 57 methylated regions of the genome in Hi-C experiments36. Similar, and perhaps equivalent, higher-order chromatin structures, referred to as Polycomb bodies137, are known to be mediated by PRC1 components in both plant and animal cells156-159. Our group has previously shown that SET-7, the catalytic component of PRC2, is required for normal 3D genome organization58. It would be interesting to learn if EPR-1, the only known effector of H3K27 methylation in N. crassa, is also essential for this wild-type chromatin organization. Despite the loss of transcriptional silencing observed in epr-1 mutants, they do not exhibit appreciably altered H3K27 methylation – this is striking for a few reasons. First, it suggests that H3K27 methylation-mediated silencing is a unidirectional pathway in N. crassa, in which EPR-1 acts downstream of PRC2. This is in contrast to findings in plants and animals, in which PRC1 components can affect the recruitment or activity of PRC2136,160,161, a fact that has hampered the elucidation of the direct role of PRC components in gene repression. Second, since ∆epr-1 strains de-repress H3K27- methylated genes without loss of H3K27 methylation, it suggests that active transcription does not necessarily preclude PRC2 activity. This was surprising since transcriptional shut-off is thought to precede PRC2 activity during normal animal development41 and because artificial gene repression can be sufficient to recruit PRC284,85. Finally, the presence of H3K27 methylation on de-repressed genes in ∆epr-1 strains demonstrates that H3K27 methylation per se is not sufficient for effective gene repression, consistent with previous reports162,163. It is noteworthy, however, that while ∆set-7 and ∆epr-1 strains share transcriptional and sexual defects, the phenotype of ∆set-7 strains is 58 generally more pronounced. This suggests that PRC2 or H3K27 methylation may have additional roles in gene repression that go beyond recruitment of EPR-1 to chromatin. Our investigation into the phylogenetic distribution of EPR-1 homologs indicates that an ancestral EPR-1 emerged prior to the divergence of plants, animals, and fungi. This ancestral EPR-1 may have been an integral component of an early eukaryotic Polycomb silencing system. While animals are a notable exception, regarding the absence of EPR-1 homologs with a BAH-PHD structure, a human BAH domain-containing protein, BAHD1, has been reported to ‘read’ H3K27me3147 and promote gene silencing164, although apparently not interact with known PRC1 components165. It is therefore conceivable that BAHD1 homologs present in animal lineages are actually divergent orthologs of EPR-1 that lack a PHD finger. Regardless of their ancestry, both human BAHD1 and N. crassa EPR-1 represent novel forms of H3K27 methylation- mediated repression that do not rely on PRC1 components. Materials and Methods Strains, media and growth conditions All N. crassa strains used in this study are listed in Appendix B. Liquid cultures were grown with shaking at 32 °C in Vogel’s minimal medium (VMM) with 1.5% sucrose29. Crosses were performed at 25 °C on modified Vogel’s with 0.1% sucrose30. Spot tests were performed at 32 °C on VMM with 0.8% sorbose, 0.2% fructose, and 0.2% glucose (FGS). When appropriate, plates included 200 µg/mL Hygromycin B Gold (InvivoGen) or 133 µg/mL Nourseothricin (Gold Biotechnology). Linear growth rates 59 were determined as previously described except 25 mL serological pipettes were used in place of glass tubes140. Genomic DNA was isolated as previously described166. Nutritional supplements required for auxotrophic strains were included in all growth media when necessary. Selection for mutants defective in H3K27 methylation-mediated silencing Ten thousand conidia of strain N6279 (created with primers in Appendix C) were plated on VMM supplemented with FGS and 500 µg/mL histidine and subjected to 0, 3, 6, or 9 seconds of ultraviolet light (Spectrolinker XL-1500 UV Crosslinker, Spectronics Corporation) in a dark room and plates were wrapped in aluminum foil to prevent photoreactivation167. Plates were incubated at 32 °C for 16 hours before being overlaid with 1% top agar containing VMM, FGS, 500 µg/mL histidine, Hygromycin B and Nourseothricin. Drug-resistant colonies were picked after an additional 48-72 hours at 32 °C. Initial mutant strains were crossed to a Sad-1 mutant168 strain (N3756) and resultant progeny were germinated on Hygromycin B- and Nourseothricin-containing medium to obtain homokaryotic mutants. Whole genome sequencing, mapping and identification of epr-1 alleles Homokaryotic mutants resistant to Hygromycin B and Nourseothricin were crossed to the genetically polymorphic Mauriceville strain (FGSC 2225)107 and resultant progeny were germinated on medium containing Hygromycin B and/or Nourseothricin to select for strains bearing the causative mutation. Genomic DNA from approximately 15- 20 progeny per mutant were pooled and sequencing libraries were prepared with a 60 Nextera kit (Illumina, FC-121-1030). All whole genome sequencing data is available on NCBI SRA (accession #PRJNA526508). To map the approximate location of a particular causative mutation, the fraction of Oak Ridge (versus Mauriceville) SNPs across the N. crassa genome was determined as previously described48 and visualized as a moving average (window size = 10 SNPs, step size = 1 SNP) with Matplotlib169. We utilized FreeBayes and VCFtools to identify genetic variants present in our pooled mutant genomic DNA but absent in the original mutagenized strain (N6279) and the Mauriceville strain118,119. Only genetic variants of high probability that were consistent with the mapping data were considered further. Western blotting N. crassa tissue from a 16 hour liquid culture of germinated conidia was collected by filtration, washed with 1x phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.5), and suspended in 500 μL of ice-cold lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 0.02-0.2% NP-40, 1 mM EDTA) supplemented with 1x HaltTM Protease Inhibitor Cocktail (Thermo Scientific). Tissue was sonicated (Branson Sonifier-450) for three sets of 10 pulses (Output = 2, Duty cycle = 80), keeping the sample on ice between sets. Insoluble material was pelleted by centrifugation at 14,000 RPM at 4 °C for 10 minutes and the supernatant used as the western sample. Anti-H3K27me3 (Cell Signaling Technology, 9733) and anti-hH3 (Abcam, ab1791) primary antibodies were used with IRDye 680RD goat anti-rabbit secondary (LI-COR, 926-68071). Anti-GFP (Thermo Fisher, A10262) primary antibody was used with goat anti-chicken HRP conjugated secondary antibody (Abcam, 6877). 61 Images were acquired with an Odyssey Fc Imaging System (LI-COR) and analyzed with Image Studio software (LI-COR). Chromatin immunoprecipitation Liquid cultures were grown for approximately 18 hours with shaking at 32 °C. Tissue samples for H3K27me2/3 ChIP were cross-linked with 0.5% formaldehyde for 10 minutes and GFP ChIP samples were cross-linked with 1% formaldehyde for 10 minutes in 1x PBS. Cross-linking was quenched with 125mM glycine, tissue was washed with 1x PBS and collected. Cells were lysed in ChIP lysis buffer (50 mM HEPES [pH 7.5], 90 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Deoxycholate) supplemented with 1x Halt protease inhibitor cocktail (Thermo Scientific) using a Branson Sonifier 450. Chromatin was sheared using a Bioruptor (Diagenode) and 2 µl of appropriate antibody (H3K27me2/3, Active Motif 39536, or GFP, MBL 598) was added and incubated with rotation at 4 °C overnight. Protein A/G agarose (40 µL; Santa Cruz Biotechnologies) was added to H3K27me2/3 ChIP samples and Protein A agarose (40 µL; Sigma) was added to GFP ChIP samples and incubated for 3 hours, rotating at 4 °C. Beads were washed twice with ChIP lysis buffer supplemented with 140 mM NaCl, once with ChIP lysis buffer with 0.5 M NaCl, once with LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and once with TE (10 mM Tris-HCl, 1 mM EDTA), all rotating at 4 °C for 10 minutes each. DNA was eluted from beads by incubation in TES (10 mM Tris-HCl, 1 mM EDTA, 1% sodium dodecyl sulfate) at 65 °C. Crosslinking was reversed by incubation at 65 °C for 16 hours and then samples were treated with proteinase K for 2 hours at 50 °C. DNA was purified using Minelute 62 columns (Qiagen) and subsequently used for qPCR with the PerfCTa SYBR Green FastMix (QuantBio, 95071-012) on a Step One Plus Real Time PCR System (Life Technologies) using primer pairs in Appendix C, or prepared for sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs). ChIP-seq data are available on the NCBI GEO database (GSE128317). ChIP-seq mapping and analysis The suite of tools available on the open-source platform Galaxy170 was used to map ChIP-sequencing reads93 against the corrected N. crassa OR74A (NC12) genome36 and to create bigWig coverage files normalized to reads per kilobase per million mapped reads (RPKM)171. ChIP-seq tracks were visualized with the Integrative Genomics Viewer96. MAnorm was used to compare H3K27me2/3 ChIP-seq coverage on all genes (designated as ‘peaks’) between samples and data were visualized with Matplotlib169. Genes were scored by their normalized H3K27me2/3 ChIP-seq coverage in wild type and the top-ranking genes (873) were designated as H3K27 methylated. RNA isolation, mRNA-seq library prep, and RT-qPCR RNA was extracted from germinated conidia grown for 16-18 hours with a 1:1:1 glass beads, NETS (300mM NaCl, 1mM EDTA, 10mM Tris-HCl, 0.2% SDS), acid phenol:chloroform mixture (5:1; [pH 4.5]) using a bead beater and ethanol precipitated. RNA was treated with DNAse I (Amplification grade; Thermo Fisher Scientific). DNAse I-treated RNA was used for RNA-seq library preparation as previously described58 or cDNA was synthesized using the SuperScript III First Strand-Synthesis System (Thermo 63 Fisher Scientific) with poly-dT primers. cDNA was used for qPCR using the PerfCTa SYBR Green FastMix (QuantBio) on a Step One Plus Real Time PCR System (Life Technologies) using primer pairs in Appendix C. mRNA-seq data are available on the NCBI GEO database (GSE128317). mRNA-seq mapping and analysis Tools available on Galaxy170 were used to map mRNA-sequencing reads (intron size < 1kb)172 against the corrected N. crassa OR74A (NC12) genome36, to count the number of reads per gene172 and to identify differentially expressed genes173. P values reported are adjusted for false discovery rates173. The Chi-squared test was used to determine if upregulated genes in ∆epr-1 strains were enriched for genes marked with H3K27 methylation. Significance of gene set intersections were calculated with a hypergeometric distribution (http://nemates.org/MA/progs/overlap_stats_prog.html). DamID Southern hybridizations and sequencing Southern hybridizations were carried out as previously described98, except probes were made with PCR products amplified from wild-type N. crassa genomic DNA (NCU05173, Tel VIIL) or plasmid pBM61 (his-3) using primer pairs from Appendix C. Preparation of N6-methyladenine-containing DNA for sequencing was performed using a previously reported procedure150 using primers in Appendix C with the following modifications: 5 μg of genomic DNA from N. crassa strains expressing a Dam fusion was digested with 1 μL of DpnI (NEB, 20 units/μL); ligation to primer 5050 was carried out overnight at 16 °C; amplification reactions of ligated DNA with primer 5051 were 64 performed in triplicate with 5 μL dNTPs, and an additional PCR cycle was added to the 2nd and 3rd phase of the PCR protocol (4 and 18 cycles respectively); 3 μg of pooled, amplified DNA was sheared using a Bioruptor (Diagenode) twice on high for 10 minutes (30 seconds on/off) at 4 °C; biotinylated DNA was purified using 250 μL slurry of streptavidin-agarose beads (Sigma). DNA was cleaved from the beads with DpnII and libraries were prepared for sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs). False perithecia assay and image analysis To assay of the development of false perithecia, strains were grown on modified Vogel’s media30 as described above without a fertilizing strain. Images of plates were acquired after two weeks of growth at 25 °C and false perithecia were detected and quantified using the Laplacian of Gaussian blob detection algorithm from scikit-image174. Microscopy image acquisition and analysis Live conidia were suspended in water and placed on a poly-L-lysine (Sigma) coated coverslip (No. 1.5; VWR) and mounted on a glass slide. Single plane images for distance measurements were captured with the ELYRA S.1 system (Zeiss) mounted on an AXIO Observer Z1 inverted microscope stand (Zeiss) equipped with a 63x, 1.4 NA Plan-Apochromat oil-immersion lens (Zeiss) and analyzed using Imaris (version 9.2.1). Images for volume renderings and max projections were collected with the DeltaVision Ultra microscope system (GE) equipped with a 100x, 1.4 NA UPlanSApo objective (Olympus). Three-dimensional Z-stack wide-field fluorescent (eGFP, TagRFP, iRFP, and 65 TagBFP) images were captured with an sCMOS camera controlled with Acquire Ultra software. Images were processed using 10 cycles of enhanced ratio deconvolution and max projections were made using softWoRx (GE, version 7.1.0). Imaris (version 9.2.1) was used to make volume renderings and TFR1 and EPR1 foci were counted by hand. Cloning, expression and purification of BAH domain from EPR-1 The BAH domain of EPR-1 (amino acids 134-264) was PCR-amplified from a plasmid containing epr-1 cDNA with primers 6681 and 6682 (Appendix C), and cloned into pE-SUMO (LifeSensors), resulting in plasmid 3410 (Appendix D). A HIS-SUMO- only control construct was generated by adding a stop codon immediately before the BAHEPR-1 domain in plasmid 3410. The HIS-SUMO-BAHEPR-1 fusion and HIS-SUMO tag constructs were expressed in Escherichia coli BL21 DE3 cells (New England BioLabs). Both HIS-SUMO-BAHEPR-1 and HIS-SUMO cultures were initially grown in 4 liters of LB media at 37 °C at 200 RPM until cultures reached an optical density of ~ 1.0 at 600 nm and then cultures were pelleted. Cells were then re-suspended separately in 1 liter of M9 medium supplemented with 15N-NH4Cl. HIS-SUMO-BAHEPR-1 cultures were induced with 0.4 mM IPTG and grown for 16-18 hours at 18 °C. HIS-SUMO cultures were induced with 1 mM IPTG and grown for 16-18 hours at 25 °C. Both cultures were collected separately by centrifugation at 6,000 RPM for 20 minutes, frozen in liquid N2, and stored at -80 °C. The same protocol outlined below was used for the purification of both HIS-SUMO-BAHEPR-1 and HIS-SUMO alone. Cells were re-suspended in lysis buffer (20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 5 mM imidazole) with DNase I, EDTA-free protease inhibitors (Roche mini- 66 tablets) and lysozyme. The cell suspension was sonicated over a period of 1 minute, alternating 1 second on and 2 seconds off. The lysate was cleared by centrifugation at 15,000 RPM for one hour at 4 °C, and the soluble fraction was loaded onto a column packed with Ni(II)-nitrilotriacetic acid agarose (Qiagen) pre-equilibrated in lysis buffer. The column was washed with 100 mL of elution buffer containing 5 mM imidazole, and the protein was eluted with buffer containing 100 mM - 200 mM imidazole. Elution fractions were then analyzed using SDS-PAGE, were pooled and concentrated using 10,000 MWCO membrane (Millipore). The protein was further purified using fast protein liquid chromatography (FPLC). Concentrated protein was loaded onto a pre-equilibrated Superdex 75 (GE Healthcare) column containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT. Size exclusion fractions were analyzed using SDS-PAGE, concentrated, and stored at -80 °C. NMR spectroscopy H3K27me3 (amino acids 23–34) peptide was obtained from Anaspec. For NMR studies, peptides were re-suspended in H2O to a final concentration of 20 mM, and pH was adjusted to 7.0. Titration experiments of peptide into HIS-SUMO-BAHEPR-1 were carried out by collecting 1H-15N HSQC spectra on 15N labeled protein at 0.09 mM in the presence of increasing peptide concentrations. Titration points were taken at protein:peptide molar ratios of 1:0, 1:1, 1:5 and 1:10. In addition, an 1H-15N HSQC was collected on 15N labeled 0.1 mM HIS-SUMO tag alone. All NMR data were collected on a Bruker Avance II 800 MHz NMR spectrometer equipped with a cryoprobe. Data were processed using NMRPipe, and further analyzed using CcpNmr. 67 Bioinformatic identification of EPR-1 and SET-7 homologs PSI-BLAST175 was used to initiate sequence similarity searches with representative sequences of EPR-1 (accession number XP_965052.2) and SET-7 (accession number XP_965043.2, residues 577-833) from N. crassa against the National Center for Biotechnology Information (NCBI) non-redundant (NR) database and locally maintained databases of proteins from representative species from different branches of the tree of life. HHpred176 was used to perform profile-profile comparisons against the Protein Data Bank (PDB), Pfam and locally maintained protein sequence profiles. Sequences were clustered using BLASTCLUST (ftp://ftp.ncbi.nih.gov/blast/documents/blastclust.html). Multiple sequence alignments were generated using Kalign177 and then adjusted manually. FastTree178 was used to assess phylogenetic relationships among the proteins retrieved after sequence similarity searches. Customized PERL scripts were used for the analysis of domain architectures and other contextual information about protein sequences. Stringent criteria of domain- architectural concordance were used to retrieve only orthologs of all proteins under study along with grouping in phylogenetic tree analysis. In the case of SET-7, only those protein sequences that have a complete pre-SET domain followed by a SET domain were considered for analysis. Replacement of NCU05173 and NCU07152 ORFs with hph and nat-1 To delete NCU07152, the 5’ and 3’ regions flanking the open reading frame (ORF) were amplified from wild-type genomic DNA with primers 6385-6388 (Appendix 68 C). The nat-1 gene was amplified from plasmid 3237 with primers 6269 and 6270. The resulting three pieces of DNA were stitched by overlap extension using primers 6385 and 6388 and the final product was transformed into N4840. Primary transformants were selected on Nourseothricin-containing medium and crossed to a wild-type strain (N3753) to remove ∆set-7 and ∆mus-52 from the genetic background, resulting in strain N5808. To delete NCU05173, the 5’ and 3’ regions flanking the ORF were amplified from wild- type genomic DNA with primers 6605-6608. The hph gene was amplified from plasmid 2283. The 5’ flank was stitched to the 5’ portion of hph by overlap extension using primers 6605 and 2955, and the 3’ flank was stitched to the 3’ portion of hph using primers 6608 and 2954. The resulting two pieces of ‘split marker’ DNA were transformed into strain N4840. Primary transformants were selected on Hygromycin B- containing medium and crossed to N5808 to generate a homokaryotic strain with both marker genes (N6233). N6233 was crossed to N623 to introduce his-3, resulting in the strain used for the mutant hunt (N6279). Creation and targeting of his-3+::pCCG::N-GFP::EPR-1 plasmids The ORF and 3’ UTR of epr-1 were PCR-amplified from wild-type genomic DNA with primers 6416 and 6417 (Appendix C) and cloned into plasmid 2406120 using PacI and XbaI restriction sites. For the BAH point mutant, epr-1W184A, two PCR products amplified from wild-type genomic DNA with primers pairs 6416 and 6368, and 6367 and 6417 were PCR-stitched together with primers 6416 and 6417, and similarly cloned into plasmid 2406. For the PHD point mutant, epr-1W292A, two PCR products amplified from wild-type genomic DNA with primer pairs 6416 and 6400, and 6399 and 6417 were 69 PCR-stitched together with primers 6416 and 6417, and cloned into plasmid 2406. Plasmids were linearized with NdeI and targeted to his-3 in either N7451 (for complementation) or N7567 (for microscopy), as previously described91. Primary transformants were then crossed to N7549 or N7552, respectively. Replacement of epr-1 with trpC::nat-1 The 5’ and 3’ flanks of epr-1 were PCR-amplified from wild-type genomic DNA with primer pairs 6401 and 6402, and 6350 and 6351, respectively (Appendix C). The 5’ and 3’ flanks were separately PCR-stitched with plasmid 3237 (source of nat-1) using primer pairs 6401 and 4883, and 4882 and 6351, respectively. These two ‘split-marker’ PCR products were transformed into strain N7537 and epr-1 replacements were selected on Nourseothricin-containing medium. Primary transformants were crossed to N3752 (to generate N7567) and N6234 (to generate N7576). Endogenous C-terminal-tagging of EPR-1 with 10xGly::Dam The regions immediately upstream (5’) and downstream (3’) of epr-1’s stop codon were PCR-amplified from wild-type genomic DNA with primer pairs 6348 and 6349, and 6350 and 6351, respectively (Appendix C). The 5’ and 3’ regions were separately PCR-stitched with plasmid 3131 (source of 10xGly::Dam::trpC::nat-1) using primer pairs 6348 and 4883, and 4882 and 6351, respectively. These two ‘split-marker’ PCR products were transformed into N2718 and ‘knock-ins’ were selected on Nourseothricin-containing medium. Primary transformants were crossed to N3752 to obtain homokaryons. To make epr-1W292A fusions with Dam, the same approach was 70 taken except the 5’ region was a PCR product stitched from two PCR products amplified from wild-type genomic DNA with primer pairs 6346 and 6400, and 6399 and 6349. 71 CHAPTER VI CONCLUDING SUMMARY In Chapter II, I investigated the role of Neurospora SIR2 homologues in the establishment of H3K27 methylation. Deletion of nst-1,4,5 (neurospora sir two-1,4,5) had minimal to no effect on the distribution of H3K27 methylation, but deletion of nst-3 unexpectedly resulted in accumulation of H3K27 methylation at a subset of centromeres. Since NST-3 is a putative H3K56 deacetylase, I wondered if this effect was mediated by hyper H3K56 acetylation (H3K56ac). Double mutants with ∆nst-3 that either lacked lysine 56 on histone H3 (H3K56R) or lacked the putative H3K56 acetyltransferase (rtt109) fully suppressed the ectopic, centromeric H3K27 methylation. Centromeric H3K27 methylation observed in strains lacking HP112 was also partially suppressed when combined with H3K56R. Finally, Vincent Bicocca looked at the distribution of H3K56ac in wild-type and ∆nst-3 strains. While he did detect hyper H3K56ac at centromeres in ∆nst-3 strains that was fully suppressed by H3K56R, the residual signal in strains bearing H3K56R suggests that the antibody used is not specific to H3K56ac. These results implicate hyper H3K56ac in ectopic accumulation of H3K27 methylation. The fact that the redistribution of H3K27 methylation in strains lacking HP1 appears to be partially suppressed by H3K56R suggests that the ectopic H3K27 methylation in ∆hpo and ∆nst-3 strains occurs through a shared pathway. However, the milder phenotype observed in ∆nst-3 strains, compared to ∆hpo strains, suggests that additional, parallel pathways promoting ectopic H3K27 methylation may also be triggered. My speculation is that hyper H3K56ac per se is not sufficient for accumulation 72 of H3K27 methylation at centromeres. I think that downstream effectors, components of an activated DNA damage response machinery, mediate the ectopic H3K27 methylation. In wild type strains, ectopic H3K27 methylation at sites of perceived DNA damage may be beneficial to block local transcription28. But, in the case of ∆hpo strains, in which all constitutive heterochromatin appears damaged, the aberrant activity of PRC2 is toxic. In Chapter III, I collaborated with Kirsty Jamieson and others to determine the effect of chromosome position on H3K27 methylation. We uncovered two classes of H3K27 methylation by examining the distribution of H3K27 methylation in strains bearing chromosomal rearrangement: position-dependent and position-independent. We found that proximity to a chromosome end was necessary to maintain and sufficient to induce subtelomeric H3K27 methylation, and that the observed changes in H3K27 methylation were reflected in altered gene expression. I further demonstrated that subtelomeric H3K27 methylation required telomerase and that ectopic telomere repeats (TTAGGG)n can induce large domains of H3K27 methylation. To the best of our knowledge, telomere repeats represent the first known cis- acting signal that direct H3K27 methylation in fungi. However, the mechanism by which these sequences induce H3K27 methylation, and not just locally, is unresolved. One can imagine that PRC2 is directly recruited to these sequences, either through an interaction with DNA, or a transcribed RNA. Alternatively, PRC2 could be recruited indirectly through another protein complex. Evidence in mammals73 and plants111 suggest the former and latter, respectively. In addition to the identification of telomere repeats as a potent signal for H3K27 methylation, our chromosomal rearrangement strains elucidated 73 regions of the N. crassa genome that are ripe for dissection to further uncover cis-acting signals. Indeed, much of the H3K27 methylation in N. crassa is telomere-independent. In Chapter IV, I collaborated with Elizabeth Wiles to identify factors required for H3K27 methylation-mediated silencing using a forward genetic selection that she implemented. We identified an undescribed gene (NCU04278) that is necessary for subtelomeric H3K27 methylation and silencing of underlying genes. Immunoprecipitation followed by mass spectrometry analysis of NCU04278-associated proteins demonstrated that NCU04278 is PRC2 complex member, and we therefore deemed it PRC2 accessory subunit (PAS). Phylogenetic analysis of PAS homologs demonstrated that they are likely limited to the fungal class Sordariomycetes and Leotiomycetes. These results help elucidate the molecular mechanism that differentiates the two classes of H3K27 methylation uncovered in Chapter III. PAS, an accessory subunit of PRC2, is responsible for the establishment of H3K27 methylation that is dependent on telomere repeats. How exactly PAS recruits or activates PRC2 specifically at subtelomeric regions is unclear. However, work with mammalian PRC2 has demonstrated that recruitment to chromatin is dependent on the N-terminus of the PRC2 member SUZ12, and that SUZ12 directly interacts with PRC2 accessory subunits that are essential for targeting116. Yeast two-hybrid experiments that examine the possible interaction between PAS and SUZ12, and subsequent in vivo targeted deletions of SUZ12 will elucidate whether the general mechanism of PRC2 targeting is conserved between animals and fungi. 74 In Chapter V, I further collaborated with Elizabeth Wiles and others, using a forward genetic selection that she implemented, to identify factors required for H3K27 methylation-mediated silencing. In this case, we found a mutation in an undescribed gene (NCU07505), whose deletion resulted in transcriptional and developmental defects shared with strains lacking all H3K27 methylation, i.e. ∆set-7, yet H3K27 methylation in ∆NCU07505 strains was essentially normal. We determined that EPR-1 (NCU07505) formed nuclear foci and localized to H3K27-methylated chromatin dependent on its BAH domain. Further in vitro work demonstrated that the BAH domain of EPR-1 directly interacts with H3K27me3 peptides. Finally, we demonstrated that homologs of EPR-1 exist in a variety of eukaryotic lineages, suggesting an ancient origin. These results fill in a major gap of our understanding regarding the mechanism of Polycomb silencing in fungi and beyond. Prior to the identification of EPR-1 in N. crassa, it was mysterious as to how H3K27 methylation could be repressive when canonical PRC1 components were absent7. Choanoflagellates, the closest living relatives to animals, also lack canonical PRC1 members7, but our phylogenetic analyses demonstrate that they have an EPR-1 homolog. In plants, EPR-1 paralogs EBS and SHL interact with PRC1 components136, but whether EPR-1 forms a complex in other lineages that lack PRC1 components is unresolved. Future biochemical work may help resolve the mechanism of silencing enacted by EPR-1 in N. crassa and beyond. 75 APPENDIX A FIGURES Figures for Chapter II Fig. 1. Aberrant H3K56ac implicated in ectopic H3K27me2/3. (A-C) ChIP-seq tracks of indicated histone modification distribution in indicated genotypes on linkage group VI. Grey bar represents 500 kilobases. 76 Figures for Chapter III Fig. 2. Chromosomal rearrangements are associated with altered H3K27me2/3. (A- F) Schematics show the movement (magenta curved arrows) of translocated segments and the resulting chromosomal rearrangements for six translocation strains. ChIPs were done on biological triplicates and pooled for sequencing. Blue and dashed lines distinguish source of chromosome while arrowheads indicate directionality of rearranged segments. H3K27me2/3 ChIP-seq tracks of WT and translocation strains are displayed above chromosome diagrams with zoom-in sections in boxes. H3K27me2/3 signals that were lost in translocation strains are shown in orange, H3K27me2/3 signals gained are indicated in green and invariant H3K27me2/3 signals are shown in black. Circles indicate centromeres. Gains and losses of H3K27me2/3 were confirmed by qPCR (Fig. 6). 77 Fig. 3. Three kilobase segments from a natural H3K27me2/3 domain are insufficient to trigger de novo H3K27me2/3 at ectopic loci. (A) Distribution of H3K27me2/3 (black), genes (gray) and H3K9me3 (blue, inverted) are displayed for the left subtelomere of LG VI. The red bar indicates the region deleted for the transplantation experiments. The eight three kb segments (expanded below the black bar) were targeted to the his-3 and csr-1 loci. Green vertical bars represent the locations of qPCR primer pairs used in H3K27me2/3 ChIP experiments. H3K27me2/3 ChIP-qPCR indicates the absence of ectopic H3K27me2/3 in strains containing regions 1-8, each separately targeted to (B) his-3 and (C) csr-1. Each ChIP experiment was performed in biological triplicate; qPCR analyses were performed on each sample in technical triplicate. Bars represent means of biological triplicates and error bars show standard deviation. 78 Fig. 4. H3K27me2/3 profiles of additional strains containing chromosomal rearrangements. Chromosome rearrangements and H3K27me2/3 ChIP-seq (pooled biological triplicates) profiles are shown for translocations UK2-32 (A), AR16 (B) and OY337 (C). Magenta curved arrows illustrate the movement of chromosomal segments in the translocations. Blue and dashed lines distinguish chromosome sources while arrows indicate the directionality of translocated segments. Light gray lines highlight regions of uncertainty with respect to the locations of breakpoints based on available genetic evidence. Solid red line indicates chromosome deletions. H3K27me2/3 ChIP-seq tracks of WT and translocation strains are displayed above chromosome diagrams. H3K27me2/3 that was lost in a translocation strain is highlighted in orange in the corresponding WT track while new H3K27me2/3 is indicated in green. Conserved H3K27me2/3 is shown in black. Circles indicate centromeres. Dark gray bars indicate 500 kb. ________________________________________________________________________ Fig. 5 (see next page). Whole genome view of H3K27me2/3 ChIP-seq in wild-type and chromosomal translocation strains. H3K27me2/3 ChIP-seq for all seven N. crassa chromosomes is displayed for WT and nine chromosomal translocation strains mapped onto the WT genome. Genes are indicated below the ChIP-seq track as vertical black bars. Grey bar represents 500 kb. 79 80 Fig. 6. qPCR validation of H3K27me2/3 ChIP-seq data from translocation strains. The gains and losses of H3K27me2/3 in ChIP-seq experiments were confirmed by ChIP- qPCR at representative regions for UK3-41 (A), OY350 (B), ALS159 (C), OY329 (D), NM149 (E), OY337 (F), UK2-32 (G), and AR16 (H). Each ChIP experiment was performed in biological triplicate; qPCR analyses were performed on each sample in technical triplicate. Bars represent means of biological triplicates and error bars show standard deviation. 81 Fig. 7. A segmental duplication confirms H3K27me2/3 position effect. (A) Diagram of a cross between the OY329 (Oak Ridge) insertional translocation strain and a polymorphic wild-type strain (Mauriceville), resulting in a strain bearing a duplicated chromosomal segment (outlined by rectangles). H3K27me2/3 ChIP-seq tracks (single experiment) and chromosomes are shown in black for the Oak Ridge strain and blue for Mauriceville. Solid and dashed lines indicate chromosome source. (B) Expanded view of the duplicated chromosome segment in parental strains and duplication-containing offspring along with associated SNP-parsed H3K27me2/3 profiles. 82 Fig. 8. Altered gene expression reflects changes in H3K27me2/3. (A) Reduced gene expression in novel subtelomeric region (LG VIL) that gains H3K27me2/3 in translocation OY350. The top panel displays H3K27me2/3 ChIP-seq reads of OY350 (green) normalized to the corresponding the non-translocated segment in WT (black) using a 350 bp sliding window. The bottom panel shows normalized mRNA-seq read counts (counts/1000) for genes (gray arrows) in the region for WT (black) and OY350 (green). ChIP-seq data are from pooled biological triplicates; RNA-seq data are from biological duplicates (B) Shifting a normally subtelomeric region to an internal position in translocation OY350 results in loss of H3K27me2/3 on LG IR and increased gene expression. The top panel displays H3K27me2/3 ChIP-seq reads of OY350 (orange) normalized to WT (black) using a 500 bp sliding window. The bottom panel shows normalized mRNA-seq read counts (counts/1000) for genes (gray arrows) in the region for WT (black) and OY350 (orange). (C) A box plot summarizes the relationship between gain (green) or loss (orange) of H3K27me2/3 in seven chromosomal translocation strains (ALS159, AR16, OY329, OY337, OY350, UK2-32, and UK3-41) and associated gene expression changes (log2[translocation/WT]) within the corresponding domains. The control (gray) represents gene expression in regions that do not exhibit changes in H3K27me2/3. 83 Fig. 9. qPCR validation of RNA-seq expression changes in translocation OY350. The effects of gain of H3K27me2/3 (A) and loss of H3K27me2/3 (B,C) on associated gene expression levels were verified by qPCR. Gene expression levels were normalized to housekeeping gene, NCU02840. qPCR analyses were performed on each sample in technical triplicate. Bars represent means of biological triplicates and error bars show standard deviation. ________________________________________________________________________ Fig. 10. (see next page) Loss of tert disrupts chromosome ends and abolishes subtelomeric H3K27me2/3. (A) Southern hybridization of genomic DNA from WT and tert strains digested with HindIII/NotI reveals loss of chromosome ends marked with the telomere repeats (TTAGGG)n, which was used as the probe. The ethidium bromide (EtBr) image demonstrates equal loading of WT and tert genomic DNA. (B) Circularization of chromosomes was demonstrated by generation of PCR products with outwardly directed primers near chromosome ends in a tert strain, but not a WT strain. (C) H3K27me2/3 ChIP-seq of tert (single sample; blue track) compared to WT (pooled biological triplicates; black track). Sequence coverage of the tert ChIP input (gray track) is also shown. 84 85 Fig. 11. Whole genome view of H3K27me2/3 ChIP-seq in wild type and tert. H3K27me2/3 ChIP-seq for all seven N. crassa chromosomes is displayed for WT (pooled biological triplicates; black track) and tert (single sample; blue). The y-axis is number of reads averaged over 25 bp windows. Open black or blue circles indicate location of centromeres. 86 Fig. 12. Telomere repeats targeted to the csr-1 locus induce an approximately 225 kb H3K27me2/3 domain. (A) qPCR analyses (biological triplicates) of H3K27me2/3 ChIP with strains containing 8 or 17 telomere repeats at the csr-1 locus, with WT strain as a control (means displayed; error bars show standard deviation; single and double asterisks represent p = 0.01488 and p = 0.00336 respectively). Level of H3K27me2/3 normalized to Telomere 1L. (B) H3K27me2/3 ChIP-seq of full WT LG I (pooled biological triplicates; black track). Black circle indicates the centromere, csr-1 position is indicated by vertical line, and arrows at chromosome ends indicate 5’ to 3’ polarity of telomere repeats. Expansion below shows the extent and shape of the H3K27me2/3 domain induced in the Δcsr-1::(TTAGGG)17 strain (single sample; green track) compared to WT (black track). Site of csr-1 replacement with telomere repeats indicated below ChIP-seq tracks. Genes are displayed as black bars. 87 Fig. 13. Telomere repeats targeted to the his-3 locus induce local H3K27me2/3 domain. qPCR analyses of H3K27me2/3 ChIP with strains containing 21, 22, or 23 telomere repeats at the his-3 locus, with WT biological triplicates as a control. Level of H3K27me2/3 normalized to Telomere 1L. 88 Fig. 14. Whole genome view of H3K27me2/3 ChIP-seq in wild type and Δcsr- 1::(TTAGGG)17. H3K27me2/3 ChIP-seq for all seven N. crassa chromosomes is displayed for WT (pooled biological triplicates) and Δcsr-1::(TTAGGG)17 (single sample). The y-axis is number of reads averaged over 25 bp windows. Open black circles indicate location of centromeres. 89 Figures for Chapter IV Fig. 15. Identification of NCU04278 as a potential component of the Polycomb silencing pathway. (A) Serial dilution spot test silencing assay for the indicated strains plated on the indicated media. All strains harbor PNCU05173::hph and PNCU07152::nat-1. (B) Whole genome sequencing of pooled hnr mutant genomic DNA identified a region on the right arm of LG V which is enriched for Oak Ridge SNPs and contained an insertional frameshift in NCU04278 (structure of which is represented as a solid gray bar, as no known domains predicted). Each translucent point represents a running average (window size = 10 SNPs, step size = 1 SNP). (C) Serial dilution spot test silencing assay for the indicated strains. hnr + NCU04278WT has a wild-type copy of NCU04278 at the his-3 locus. All strains harbor PNCU05173::hph. 90 Fig. 16. Mapping and characterization of an additional NCU04278 allele. (A) Whole genome sequencing of pooled mutant genomic DNA identified a region on the right arm of LG V which is enriched for Oak Ridge SNPs and contained a nonsense mutation in NCU04278. Each translucent point represents a running average (window size = 10 SNPs, step size = 1 SNP). (B) Diagram of the primary structure of each indicated NCU04278 allele (light gray bars are wild-type sequence; dark gray bar is frameshifted sequence). Predicted intrinsically disordered regions within NCU04278 are labelled above (red bars). 91 Fig. 17. Loss of NCU04278 upregulates a subset of SET-7 repressed genes near chromosome ends. (A) Venn diagram depicting genes (numbers in bold) that appear upregulated by mRNA-seq in both ∆NCU04278 and ∆set-7 strains, only in ∆NCU04278 strains, or only in ∆set-7 strains, using a significance cutoff of log2(mutant/wild type) > 1 and P value < 0.05. The percentage of total upregulated genes that are H3K27 methylated for each gene set is indicated below the total gene count. Significance of overlapping gene sets was determined using a hypergeometric test. (B) Same as in A, but for downregulated genes (log2(mutant/wild type) < -1). (C) Box and whisker plot of the distances from the telomere of H3K27-methylated genes that appear upregulated by mRNA-seq in the indicated genotypes. Boxes represent interquartile range, horizontal lines represent the median, and whiskers represent minimum and maximum values. (D) RT-qPCR of H3K27-methylated genes that were replaced with antibiotic resistance genes (NCU07152 and NCU05173) and used for initial selection of mutants, and H3K27- methylated genes that appeared upregulated in both ∆NCU04278 and ∆set-7 strains by mRNA-seq (NCU09633, NCU04991, NCU09604, NCU09306, NCU10070, NCU10038). Each value was normalized to expression of NCU02840 and presented relative to wild type. Filled bars represent the mean from biological triplicates and error bars show standard deviation (**** for P < 0.0001, *** for P < 0.001, ** for P < 0.01, * for P < 0.05, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test). 92 Fig. 18. Genome-wide distribution and level of H3K27 methylation in wild-type, ∆NCU04278, and ∆npf strains. (A) H3K27me2/3 ChIP-seq of merged biological replicates of wild type (black), ∆NCU04278 (red), and ∆npf (gray) strains displayed for all seven LGs of N. crassa. LG I is split at the right end of its centromere into IL and IR. Y-axes are 900, 850, and 450 RPKM for wild type, ∆NCU04278, and ∆npf respectively. Black bar represents 500 kb. (B) Scatter plot showing the correlation of H3K27me2/3 levels at all genes (dots) in wild type and ∆NCU04278 based on merged biological replicates of ChIP-seq data. Dots representing genes are colored based on their relative H3K27me2/3 levels in ∆NCU04278 compared to wild type (red for log2(∆NCU04278/wild type) > 1, gray for -1 < log2(∆NCU04278/wild type) < 1, and blue for log2(∆NCU04278/wild type) < -1). Representative genes that gained (NCU07801) or lost (NCU07152) H3K27me2/3 in ∆NCU04278 are indicated. (C) H3K27me2/3 ChIP- qPCR to validate ChIP-seq data at eight regions – NCU05086, NCU08085, NCU08251 (positive controls); NCU05173, NCU07152, Telomere IIIR (lost H3K27me2/3 in ∆NCU04278); NCU07801 (gained H3K27me2/3 in ∆NCU04278); hH4 (negative control). Filled bars represent the mean of biological triplicates and error bars show standard deviation (**** for P < 0.0001, *** for P < 0.001, * for P < 0.05, ns for not significant; all relative to wild type by two-tailed, unpaired t-test). 93 Fig. 19. The effect of loss of NCU04278 on H3K27 methylation and growth rate. (A) Box and whisker plot of distance from the telomere of genes that lost (log2(∆NCU04278/wild type) < -1, n = 284) and gained (log2(∆NCU04278/wild type) > 1, n = 34) H3K27me2/3 in ∆NCU04278. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. (B) Analysis of gene expression level of genes that gain H3K27me2/3 in ∆NCU04278 strains. (C) Linear growth rates measured by ‘race tubes’ are shown for two biological replicates of wild- type (N3752, N3753), ∆set-7 (N4718, N4730), ∆NCU04278 (N7999, N8002), and ∆npf (N8003, N8004) strains. Horizontal lines represent the mean of three technical replicates (open circles). 94 Fig. 20. NCU04278 associates with known N. crassa PRC2 members. (A) Exclusive unique peptide counts from immunoprecipitation followed by mass spectrometry (IP-MS) experiments. Each column represents the results of a single IP-MS with the indicated ‘baits.’ The 3xFLAG-EED dataset is previously published. The 3xFLAG-control is a nuclear protein that localizes to H3K27 methylated chromatin (NCU07505). (B) Protein interaction network of NCU04278-associated proteins. A separate IP-MS experiment was performed for each PAS (NCU04278), SUZ12, EED (solid blue circles) and the negative nuclear control (solid grey circle). IP-MS was not performed for SET-7 or NPF (empty blue circles). Arrows point to each ‘baits’ (solid blue circles) respective associated proteins. 95 Fig. 21. PAS homologs appear restricted to fungal lineages. Phylogenetic tree highlighting select fungal species in class Sordariomycetes (red background) or Leotiomycetes (orange background) that contain predicted PAS homologs. Results of the Basic Local Alignment Search Tool (BLAST, light blue background), using Neurospora crassa PAS as the query, for each of the indicated species, is displayed to the right of the tree. Solid colored bars indicated an aligned region. Grey lines are for visual reference. Amino acid (AA) position in PAS is indicated below the alignments. Alignments are color coded according to their alignment score (yellow background). 96 Figures for Chapter V Fig. 22 (see next page). Forward genetics identifies a novel gene, epr-1, required for H3K27 methylation-mediated repression. (A) mRNA-seq results for two genes repressed by the N. crassa H3K27 methyltransferase, encoded by set-7. (B) Selection scheme, utilizing reporter genes illustrated in A, to identify factors required for H3K27 methylation-mediated silencing. (C) Serial dilution spot test silencing assay for the indicated strains plated on the indicated media. All strains harbor PNCU05173::hph and PNCU07152::nat-1. (D) Scheme for genetic mapping of critical mutation in epr-1UV1. (E) Whole genome sequencing of pooled epr-1UV1 mutant genomic DNA identified a region on the left arm of linkage group I that is enriched for Oak Ridge single nucleotide polymorphisms (SNPs) and contains a premature stop codon in the BAH domain of NCU07505 (BAH domain, light blue; PHD finger (split), dark blue; no annotated domains, gray). Each translucent point represents a running average of SNPs (window size = 10 SNPs, step size = 1 SNP). (F) Serial dilution spot test silencing assay for the indicated strains. epr-1UV1 + NCU07505WT has a wild-type copy of NCU07505 at the his- 3 locus. All strains harbor PNCU05173::hph. 97 98 Fig. 23. Three additional alleles of epr-1 were identified during selection for mutants defective in H3K27 methylation-mediated repression. (A) Whole genome sequencing of pooled genomic DNAs of mutant progeny, isolated from three additional mutants crossed with wild-type Mauriceville, independently identified a region on the left arm of LG I that was enriched for Oak Ridge SNPs and contained mutations in epr-1 (NCU07505). (B) Domain structure of NCU07505 showing the location and nature of the mutations in the epr-1 alleles. Exons are indicated as boxes, with the BAH domain in light blue, the PHD finger (split) in dark blue, and exons with no known domains in gray. Gray lines connecting exons indicate the introns. 99 Fig. 24. ∆epr-1 and ∆set-7 strains share defects in transcriptional silencing and sexual development. (A) Venn diagram depicting H3K27-methylated genes that appear upregulated by mRNA-seq in both ∆epr-1 and ∆set-7 strains, only in ∆epr-1 strains, or only in ∆set-7 strains, using a significance cutoff of log2(mutant/wild type) > 1 and a P value < 0.05. Significance of genes upregulated in both ∆epr-1 and ∆set-7 strains was determined using a hypergeometric test. (B) RT-qPCR of H3K27-methylated genes that were replaced with antibiotic resistance genes (NCU07152, NCU05173) and used for initial selection of mutants, and H3K27-methylated genes that appeared upregulated in both ∆epr-1 and ∆set-7 strains by mRNA-seq (NCU09640, NCU07624, NCU09178, NCU08570, NCU010038, NCU08097). Each value was normalized to expression of actin gene (act) and presented relative to wild type. Filled bars represent the mean from biological triplicates and error bars show standard deviation. (*** for P < 0.001, ** for P < 0.01, * for P < 0.05, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test). (C) Quantification of false perithecia developed in a Petri dish (85 mm diameter) after two weeks of unfertilized growth are shown for the indicated strains. Horizontal lines and numbers indicate the mean of two biological replicates (open circles). 100 Fig. 25 (see next page). Gene expression and phenotypic analysis of ∆epr-1 and ∆set- 7 strains. (A) Bold numbers indicate genes upregulated (left) or downregulated (right) in only ∆epr-1 strains, only ∆set-7 strains, or in both ∆epr-1 and ∆set-7 strains by mRNAseq. Significance was determined using a cutoff of log2(mutant/wild type) > 1 for upregulated genes and < -1 for downregulated genes with a P value < 0.05. The percentage of upregulated genes that are marked by H3K27 methylation for each gene set is indicated below the gene count. (B) RT-qPCR of H3K27-methylated genes that were replaced with antibiotic resistance genes (NCU07152, NCU05173), and used for initial selection of mutants, and H3K27-methylated genes that appeared upregulated in both ∆epr-1 and ∆set-7 strains by mRNA-seq (NCU09640, NCU07624, NCU09178, NCU08570, NCU010038, NCU08097). Each value was normalized to gene expression of NCU02840 (an alternative housekeeping gene) and presented relative to wild type. (C) RT-qPCR for three genes (NCU09990, NCU07241, NCU07246) that appeared upregulated only in ∆epr-1 strains by mRNA-seq. Each value was normalized to gene expression of act (left) or NCU02840 (right) and presented relative to wild type. (D) RT- qPCR for three genes (NCU09604, NCU16720, NCU09274) that appeared upregulated only in ∆set-7 strains by mRNA-seq. Each value was normalized to gene expression of act (left) or NCU02840 (right) and presented relative to wild type. For all RT-qPCR data, filled bars represent the mean from biological triplicates and error bars show standard deviation (*** for P < 0.001, ** for P < 0.01, * for P < 0.05, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test) (E) Linear growth rates measured by ‘race tubes’ are shown for two biological replicates of wild-type (N3752, N3753), ∆set-7 (N4718, N4730), and ∆epr-1 (N7497, N7491) strains. Horizontal lines represent the mean of three technical replicates (open circles). (F) Representative images of biological replicates graphed in Fig. 24C of wild-type, ∆epr-1 and ∆set-7 strains grown unfertilized at 25 °C for two weeks on modified Vogel’s with 0.1% sucrose. Overlaid black bar represents 2 cm. 101 102 Fig. 26. epr-1 is not required for H3K27 methylation. (A) Western blot showing H3K27me3 and total histone H3 (hH3) in the indicated strains. Biological replicates are shown. The same lysate was run on separate gels, and hH3 was used as a sample processing control. (B) ChIP-seq track showing average levels of H3K27me2/3 from two biological replicates of wild-type and ∆epr-1 strains on LG VI. Open circle indicates the middle of the centromere region. Gray bar represents 500 kb. Y-axis is 0-800 RPKM for wild type and 0-1200 RPKM for ∆epr-1. (C) Scatter plot showing the correlation of H3K27me2/3 levels at all genes (black dots) in wild type and ∆epr-1 strains based on biological replicates of ChIP-seq data. Line of best fit displayed in red (R2 = 0.9105). Representative genes that gained (NCU02856) or lost (NCU08834) H3K27me2/3 in ∆epr-1 are indicated. (D) H3K27me2/3 ChIP-qPCR to confirm ChIP-seq data at six regions in wild-type, ∆set-7 and ∆epr-1 strains: hH4 (negative control), Tel IL (unchanged H3K27me2/3 in ∆epr-1), NCU07152 (unchanged H3K27me2/3 in ∆epr-1), NCU05173 (unchanged H3K27me2/3 in ∆epr-1), NCU08834 (loss of H3K27me2/3 in ∆epr-1) and NCU02856 (gain of H3K27me2/3 in ∆epr-1). Filled bars represent the mean of biological triplicates and error bars show standard deviation (** for P < 0.01, * for P < 0.05, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test). 103 Fig. 27. H3K27 methylation levels in ∆epr-1 are comparable to wild type. (A) Western blot showing H3K27me3 and total histone H3 (hH3) in the indicated strains (additional replicate of experiment illustrated in Fig. 26A). The same lysate was run on separate gels, and hH3 was used as a sample processing control. (B) Quantification of the H3K27me3 band intensity averaged from 4 biological replicates. Each band intensity is normalized to the corresponding hH3 band and to the wild-type average. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units; **** for P < 0.0001, ** for P < 0.01, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test). (C) Full blots with molecular weight markers in kilodaltons (kDa) for the cropped images in Fig. 26A and panel A (above) are shown. (D) Scatter plot showing the correlation of H3K27me2/3 levels at all genes (black dots) for the two wild-type ChIP-seq biological replicates. Line of best fit displayed in red (R2 = 0.9272). (E) Scatter plot showing the correlation of H3K27me2/3 levels at all genes (black dots) for the two ∆epr-1 ChIP-seq biological replicates. Line of best fit displayed in red (R2 = 0.8941). 104 Fig. 28. EPR-1 forms telomere-associated foci that are dependent on EED and the BAH domain of EPR-1. Maximum intensity projection images of fluorescence microscopy Z-stacks showing EPR-1 (GFP-EPR-1, green) for epr-1WT (A), epr-1WT; ∆eed (B), epr-1BAH (C), and epr-1PHD (D). Telomeres (TRF1-TagRFP-T, red), centromeres (CenH3-iRFP670, magenta), the nuclear membrane (ISH1-TagBFP2, blue), and merged images are shown for reference. Each image shows a single conidium with multiple nuclei. Overlaid white bar represents 2 µm. 105 Fig. 29. EPR-1WT and EPR-1PHD exhibit no difference in distance to telomeres or EPR-1 foci number. (A) Histogram shows the relative frequency of distances between an EPR-1WT focus and the closest TRF1 focus (black bars) or CenH3 focus (gray bars) (n = 203, P = 0.0403, two-tailed Mann-Whitney test). (B) Histogram shows the relative frequency of nuclei with the indicated number of TRF1 or EPR-1WT foci (n = 194, P = 0.7422, Wilcoxon matched-pairs test). (C) Stacked bar graph comparing the number of TRF1 and EPR-1WT foci within a single nucleus (n = 194). (D) Histogram shows the relative frequency of distances between an EPR-1WT focus and the closest TRF1 focus (black bars; n = 203) or an EPR-1PHD focus and the closest TRF1 focus (gray bars; n = 241) (P = 0.8201, two-tailed Mann-Whitney test). (E) Histogram shows the relative frequency of EPR-1 foci per nucleus for EPR-1WT (n = 194) or EPR-1PHD (n = 120) (P > 0.9999, Wilcoxon matched-pairs test). 106 Fig. 30. EPR-1 directly interacts with H3K27 methylated chromatin through its BAH domain. (A) ChIP-seq and DamID-seq tracks showing average levels from two biological replicates for the indicated genotypes on LG III. Y-axis is 0-800 RPKM for H3K9me3 and H3K27me2/3 ChIP-seq, 0-500 RPKM for all GFP ChIP-seq, and 0-3500 RPKM for all DamID-seq. Gray bar represents 500 kb. (B) GFP ChIP-qPCR to validate GFP-EPR-1 ChIP-seq data at four genomic regions: Tel IL (H3K27-methylated), NCU05173 promoter (H3K27-methylated), NCU07152 promoter (H3K27-methylated), and H3K9me3 region (negative control, LG VI centromere). All data are normalized to a negative, euchromatic control, hH4. Filled bars represent the mean and error bars show standard deviation from three biological replicates (*** for P < 0.001, ** for P < 0.01, and ns for not significant; all relative to wild type by two-tailed, unpaired t-test). (C) Serial dilution spot test silencing assay for the indicated strains. All strains harbor PNCU05173::hph and PNCU07152::nat-1. (D) Two regions of an overlay of 1H-15N HSQC spectra of 15N-labelled HIS-SUMO-BAHEPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. Spectra are color coded as indicated. 15N-labelled HIS-SUMO is included for reference. 107 Fig. 31 (see next page). EPR-1 associates with H3K27 methylation in vivo and in vitro. (A) Scatter plot showing the correlation of levels of H3K27me2/3 and GFP-EPR- 1WT for all genes (black dots), as determined by ChIP-seq. Line of best fit displayed in red (R2=0.8675). (B) Western blot shows GFP-EPR-1 expression in the indicated strains. Phosphoglycerate kinase (PGK) is used as a loading control. Each genotype (except wild type, negative control) was run in biological duplicate and repeated. (C) Quantification of the GFP band intensity averaged over 4 biological replicates. The intensities are relative to the corresponding PGK band and normalized to the wild-type average from the same blot. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units). (D) Box and whisker plot of normalized GFP-EPR-1 ChIP-seq coverage from epr-1WT, epr-1PHD and epr-1BAH strains is shown for the indicated regions of the genome. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. (E) Box and whisker plot of normalized EPR-1 DamID-seq coverage from epr-1WT, epr-1PHD and epr-1WT; ∆eed strains is shown for the indicated regions of the genome. Reads have been corrected for the frequency of GATC sites. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. (F) DamID Southern blot of genomic DNA from the indicated strains digested with DpnI (DI), DpnII (DII) or left undigested (-). DpnII, which digests GATC sites without methylated adenines, reveals pattern of complete digestion in wild type. DpnI, which fails to digest GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions (NCU05173 and Tel VIIL, H3K27- methylated; his-3, euchromatin). Ethidium bromide (EtBr) shows total DNA. Biological replicates are shown. (G) An overlay of 1H-15N HSQC spectra of 15N-labelled HIS- SUMO-BAHEPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. (H) An overlay of 15N-labelled HIS-SUMO-BAHEPR-1 fusion with the 15N- labelled HIS-SUMO alone. Boxed areas are shown enlarged in Fig. 30D. Spectra are color coded as indicated 108 109 Fig. 32. EPR-1 homologs are present in species beyond fungi. (A) The presence and absence of EPR-1 and SET-7 protein homologs across major species divisions is depicted in a representative tree of eukaryotes. The leaves of the tree are labelled with the names of the divisions. Representative species are featured and their associated squares in the EPR-1 and SET-7 columns indicate the presence or absence of homologs, as indicated. (B) Protein domain structure of EPR-1, as well as EBS and SHL from Arabidopsis thaliana (A.t.). BAH domains are indicated by light blue, the PHD fingers by dark blue, and regions with no known domains are gray. Aromatic amino acid residues involved in methylated histone recognition in the BAH domain and PHD finger are indicated above the protein structure diagram (black text). Red text above the PHD finger in EPR-1 highlights the absence of aromatic residues at these amino acid positions. 110 APPENDIX B STRAIN TABLES All strains are N. crassa. Chapter II Strain Genotype N2931 mat a; ∆mus-52::bar N3752 (FGSC 2489) mat A; Oak Ridge N4922 mat a; ∆hpo::hph N6227 mat a; ∆nst-3::nat-1 N6355 mat a; ∆nst-4::hph N6420 mat A; ∆rtt109::hph N6421 mat a; ∆nst-3::nat-1; ∆rtt109::hph N6445 mat a; H3K56R::nat-1 N6539 mat a; ∆nst-1::hph N6543 mat a; ∆nst-5::hph N6585 ∆hpo::hph; H3K56R::nat-1 N6734 ∆nst-3::nat-1; H3K56R::nat-1 Chapter III Strain Genotype N51 (FGSC 2225) mat A; Mauriceville N625 mat a; his-3 N2834 mat A; his-3; Δmus-52::hph+ N2931 mat a; Δmus-52::bar+ N3752 (FGSC 2489) mat A; Oak Ridge N4730 mat A; Δset-7::bar+ N4933 mat a; Δ47.4 kb::hph+ N5100 (FGSC 1614) mat a; In(IL->IR)AR16 N5101 (FGSC 2100) mat A; T(IV->VI)ALS159 N5102 (FGSC 3670) mat A; T(VI->III)OY329 N5547 mat a; Δmus-52::bar+; Δ47.4 kb::hph+ N5683 mat A; his-3+::1; Δ47.4 kb::hph+ N5684 mat A; his-3+::2; Δ47.4 kb::hph+ N5685 mat A; his-3+::3; Δ47.4 kb::hph+ N5686 mat A; his-3+::4; Δ47.4 kb::hph+ N5687 mat A; his-3+::5; Δ47.4 kb::hph+ N5688 mat A; his-3+::6; Δ47.4 kb::hph+ N5689 mat A; his-3+::7 Δ47.4 kb::hph+ N5690 mat A; his-3+::8; Δ47.4 kb::hph++ 111 N5695 mat a; Δcsr-1::1; Δ47.4 kb::hph+ N5696 mat a; Δcsr-1::2; Δ47.4 kb::hph+ N5697 mat a; Δcsr-1::3; Δ47.4 kb::hph+ N5698 mat A; Δcsr-1::4; Δ47.4 kb::hph+ N5699 mat A; Δcsr-1::5; Δ47.4 kb::hph+ N5700 mat a; Δcsr-1::6; Δ47.4 kb::hph+ N5701 mat a; Δcsr-1::7; Δ47.4 kb::hph+ N5702 mat A; Δcsr-1::8; Δ47.4 kb::hph+ N5739 mat A; his-3; Δ47.4 kb::hph+ N5857 (FGSC 1483) mat A; T(II->V)NM149 N5858 (FGSC 3668) mat A; T(II->IV)OY337; al-2 N5859 (FGSC 7294) mat A; T(IVR->VL)UK2-32 N5862 (FGSC 4641) mat A; T(VIL->IR)OY350 N5863 (FGSC 3635) mat A; T(VI->III)OY320 N5866 (FGSC 6869) mat A; inl; T(VR )UK3-41 N6089 mat a; Dp(VI->III)OY329 N6093 (FGSC 3671) mat a; T(VI->III)OY329 N6228 mat A; Δtert::nat-1+ N6381 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)8 N6383 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)17 N6984 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)8 N6985 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)8 N6986 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)17 N6987 mat a; Δmus-52::bar+; Δcsr-1::(TTAGGG)17 N7515 mat A; Δmus-52::hph+; his-3+::(TTAGGG)23 (heterokaryon) N7516 mat A; Δmus-52::hph+; his-3+::(TTAGGG)22 (heterokaryon) N7517 mat A; Δmus-52::hph+; his-3+::(TTAGGG)21 (heterokaryon) Chapter IV Strain Genotype N51 (FGSC 2225) mat A; Mauriceville N2930 mat A; his-3; ∆mus-52::bar N3752 (FGSC 2489) mat A; Oak Ridge N3753 (FGSC 4200) mat a; Oak Ridge N3756 mat A; Sad-1; his-3 N4666 mat a; suz12::C-Gly::3xFLAG::hph N4718 mat a; ∆set-7::hph N4730 mat A; ∆set-7::bar N4879 mat a; hph::Pqa-2::3xFLAG::N-Gly::eed N6232 mat a; pNCU05173::hph N6279 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3 N6762 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; NCU04278UV1 112 N7600 mat A; pNCU05173::hph; pNCU07152::nat-1; ∆set-7::bar N7617 mat a; ∆NCU04278::nat-1 N7667 mat a; pNCU05173::hph; ∆NCU04278::nat-1 N7680 mat a; ∆NCU04278::nat-1 N7742 mat A; pNCU05173::hph; pNCU07152::nat-1; his-3; NCU04278UV1 N7807 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3+::Pccg-1::NCU04278WT::C-Gly::3xFLAG; NCU04278UV1 N7950 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; NCU04278UV2 N7999 mat a; ∆NCU04278::nat-1 N8002 mat A; ∆NCU04278::nat-1 N8003 mat a; ∆npf::hph N8004 mat a; ∆npf::hph Chapter V Strain Genotype N51 (FGSC 2225) mat A; Mauriceville N623 mat A; his-3 N625 mat a; his-3 N2718 mat a; ∆mus-52::hph N3752 (FGSC 2489) mat A; Oak Ridge N3753 (FGSC 4200) mat a; Oak Ridge N3756 mat A; Sad-1; his-3 N4718 mat a; ∆set-7::hph N4730 mat A; ∆set-7::bar N4840 mat A; ∆set-7::bar; ∆mus-52::bar N5808 mat A; pNCU07152::nat-1 N6223 mat A; ish-1::TagBFP2::hph; hpo::TagRFP-T::hph; cenH3::iRFP670::hph N6233 mat a; pNCU05173::hph; pNCU07152::nat-1 N6234 mat A; pNCU05173::hph; ∆set-7::bar N6279 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3 N6761 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; epr-1UV3 N7451 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; epr-1UV1 N7552 mat A; ish-1::TagBFP2::hph; cenH3::iRFP670::hph; his-3 N7567 mat a; ∆epr-1::nat-1; trf-1::TagRFP-T::hph; his-3 N7596 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; epr-1UV4 N7549 mat A; pNCU05173::hph; pNCU07152::nat-1; his-3; epr-1UV1 N7479 mat A; ∆epr-1::hph N7491 mat a; ∆epr-1::hph N7525 mat A; epr-1WT::10xGly::Dam::nat-1 N7526 mat a; epr-1WT::10xGly::Dam::nat-1 113 N7537 mat a; trf-1::TagRFP-T::hph; his-3 N7538 mat a; epr-1WT::10xGly::Dam::nat-1; ∆eed::hph N7539 mat a; epr-1WT::10xGly::Dam::nat-1; ∆eed::hph N7600 mat A; pNCU05173::hph; pNCU07152::nat-1; ∆set-7::bar N7900 mat a; epr-1W292A::10xGly::Dam::nat-1 N7901 mat a; epr-1W292A::10xGly::Dam::nat-1 N7576 mat A; ∆epr-1::nat-1; pNCU05173::hph N7689 pNCU05173::hph; pNCU07152::nat-1; his-3+::pCCG::N- GFP::epr-1WT; epr-1UV1 N7690 pNCU05173::hph; pNCU07152::nat-1; his-3+::pCCG::N- GFP::epr-1WT; epr-1UV1 N7722 mat a; ∆epr-1::nat-1; trf-1::TagRFP-T::hph; ish- 1::TagBFP2::hph; cenH3::iRFP670::hph; his-3+::pCCG::N- GFP::epr-1WT N7743 mat a; ∆epr-1::nat-1; trf-1::TagRFP-T::hph; ish- 1::TagBFP2::hph; cenH3::iRFP670::hph; his-3+::pCCG::N- GFP::epr-1WT; ∆eed::hph N7752 mat a; pNCU05173::hph; pNCU07152::nat-1; his- 3+::pCCG::N-GFP::epr-1W184A; epr-1UV1 N7753 mat A; pNCU05173::hph; pNCU07152::nat-1; his- 3+::pCCG::N-GFP::epr-1W184A; epr-1UV1 N7754 mat A; pNCU05173::hph; pNCU07152::nat-1; his- 3+::pCCG::N-GFP::epr-1W292A; epr-1UV1 N7755 mat a; pNCU05173::hph; pNCU07152::nat-1; his- 3+::pCCG::N-GFP::epr-1W292A; epr-1UV1 N7756 mat a; ∆epr-1::nat-1; trf-1::TagRFP-T::hph; ish- 1::TagBFP2::hph; cenH3::iRFP670::hph; his-3+::pCCG::N- GFP::epr-1W184A N7922 ∆epr-1::nat-1; trf-1::TagRFP-T::hph; ish-1::TagBFP2::hph; cenH3::iRFP670::hph; his-3+::pCCG::N-GFP::epr-1W292A N8039 mat a; pNCU05173::hph; pNCU07152::nat-1; his-3; epr-1UV2 114 APPENDIX C PRIMER TABLES Chapter II Primer Description Sequence (5’->3’) 3498 hH3_5FP CCCAATAAGTTCAAACCG 4883 nat-1_RP AACCCCATCCGCCGGTACGCG 4884 nat-1_FP TCCTTCACCACCGACACCGTCTTCC 5303 hH3K56R_FP GAGCAGCTCAGTGGACCTCTGGTATCTCCTGAT 5304 hH3K56R_RP ATCAGGAGATACCAGAGGTCCACTGAGCTGCTC 5305 hH3K56Q_FP AGCAGCTCAGTGGACTGCTGGTATCTCCTGATC 5306 hH3K56Q_RP GATCAGGAGATACCAGCAGTCCACTGAGCTGCT GTCTGCTTGGTCCTGGCCATTGTGATGGTTTATGT 5307 hH3_5RP GTGTTGATAAC GGTGAGAGAAATTAAGCGACTCTTCGATATGGAG 5333 hH3_3UTR_FP TAG CCTCCGCCTCCGCCTCCGCCGCCTCCGCCGCGGGT 5334 hH3_3UTR_RP CACAGTGATGTTTATG GAGCTCGGTACCAAGCTTGATGCATAGCTGAGAC 5335 hH3_3FP GCGTCCGTTCGC 5336 hH3_3RP GGGTTACCTTGGTTCACATGC ATCGAAGAGTCGCTTAATTTCTCTCACCACGCAGT 5337 hH3_codon_RP CTTCG Chapter III Primer Description Sequence (5’->3’) 3325 CGGGATCCCCGGATCGAACGGCGConstruction of his- 3+::1 (N5683) GATGG (BamHI) 3326 GCTCTAGATGAGCTCCTTCCCCCConstruction of his- 3+::1 (N5683) ACGGT (XbaI) 3327 CGGGATCCCGTGGGTTCCAGTGCConstruction of his- 3+::2 (N5684) GTCCC (BamHI) 3328 GCTCTAGATGGCAAGCGCCGACAConstruction of his- 3+::2 (N5684) TGTGA (XbaI) 3329 GGGCCCAGGGAACAGGCGCAGTConstruction of his- 3+::3 (N5685) GCAG (ApaI) 3330 + GACTAGTTGGCGCGCTTGACCAGConstruction of his- 3 ::3 (N5685) CAAA (SpeI) 3331 + GACTAGTTCCCGGAACGGCCACTConstruction of his- 3 ::4 (N5686) CCAT (SpeI) 3332 GCTCTAGAGTGCTGCCAAGGCCCConstruction of his- 3+::4 (N5686) GACAT (XbaI) 115 3297 Construction of his-3 +::5 (N5687) GGAATTCCAAAGGATCGCGCCCG GAGG (EcoRI) Construction of his-3+3333 ::5 (N5687) CGGGATCCACGACGGGAAGACG AGGGGT (BamHI) 3334 GGGCCCGGGAGTGTGGCGCTGTGConstruction of his- 3+::6 (N5688) GAC (ApaI) 3335 Construction of his- 3+ GACTAGTAGCAGCATCTCGGGTC ::6 (N5688) CGGT (SpeI) 3301 + GGAATTCGGCATGCCCAGGGATTConstruction of his- 3 ::7 (N5689) GGGG (EcoRI) 3336 + GCTCTAGAGACGGAACGGTGACConstruction of his- 3 ::7 (N5689) AGGCGG (XbaI) 3303 + GGAATTCTGGTTCGCGGACGAGGConstruction of his- 3 ::8 (N5690) GCTA (EcoRI) 3337 CGGGATCCGGCTATTGCTGCCAGConstruction of his- 3+::8 (N5690) CCGGT (BamHI) 4654 3’ csr-1 RP AACACCTCCGTCGCCATAAACTCC 4655 5’ csr-1 FP GGCCCCTGGTTTACTGAGGGC 4721 5’ csr-1 RP TGCAGCCATTGACGACATTGC 4722 3’ csr-1 FP TGGATTTCCTGCGCTGCACAC 4723 Construction of Δcsr-1::1 (N5695) GCAATGTCGTCAATGGCTGCACCGGATCGAACGGCGGATGG 4724 Construction of Δcsr-1::1 (N5695) GTGTGCAGCGCAGGAAATCCATGAGCTCCTTCCCCCACGGT 4725 Construction of Δcsr-1::2 (N5696) GCAATGTCGTCAATGGCTGCACG TGGGTTCCAGTGCGTCC 4726 Construction of Δcsr-1::2 (N5696) GTGTGCAGCGCAGGAAATCCATG GCAAGCGCCGACATGTGA 4727 Construction of Δcsr-1::3 (N5697) GCAATGTCGTCAATGGCTGCAAGGGAACAGGCGCAGTGCAG 4728 Construction of Δcsr-1::3 (N5697) GTGTGCAGCGCAGGAAATCCATGGCGCGCTTGACCAGCAAA 4729 Construction of Δcsr-1::4 (N5698) GCAATGTCGTCAATGGCTGCATCCCGGAACGGCCACTCCAT 4730 Construction of Δcsr-1::4 (N5698) GTGTGCAGCGCAGGAAATCCAGTGCTGCCAAGGCCCGACAT 4731 Construction of Δcsr-1::5 (N5699) GCAATGTCGTCAATGGCTGCACAAAGGATCGCGCCCGGAGG 4732 Construction of Δcsr-1::5 (N5699) GTGTGCAGCGCAGGAAATCCAACGACGGGAAGACGAGGGGT 4733 Construction of Δcsr-1::6 (N5700) GCAATGTCGTCAATGGCTGCAGGGAGTGTGGCGCTGTGGAC 4734 Construction of Δcsr-1::6 (N5700) GTGTGCAGCGCAGGAAATCCAAGCAGCATCTCGGGTCCGGT 116 4735 Construction of Δcsr-1::7 (N5701) GCAATGTCGTCAATGGCTGCAGGCATGCCCAGGGATTGGGG 4736 Construction of Δcsr-1::7 (N5701) GTGTGCAGCGCAGGAAATCCAGACGGAACGGTGACAGGCGG 4737 Construction of Δcsr-1::8 (N5702) GCAATGTCGTCAATGGCTGCATGGTTCGCGGACGAGGGCTA 4738 Construction of Δcsr-1::8 (N5702) GTGTGCAGCGCAGGAAATCCAGGCTATTGCTGCCAGCCGGT 4784 qChIP his-3 & csr-1 targeting P1Fwd AATGCAAGGTCCCGAACACT 4785 qChIP his-3 & csr-1 targeting P1Rev TGGCTGTCGCAATTACCAGT 4786 qChIP his-3 & csr-1 targeting P2Fwd GACCAAGCATGCGTTAGCTG 4787 qChIP his-3 & csr-1 targeting P2Rev ACCCAAGGTGGGTGTGTTTT 4788 qChIP his-3 & csr-1 targeting P3Fwd CCGTTTGAGCTGGTCTTCCT 4789 qChIP his-3 & csr-1 targeting P3Rev TGACGGATGCTCTTTGTCCC 4790 qChIP his-3 & csr-1 targeting P4Fwd CAACCAGCTTGACGGCTTTC 4791 qChIP his-3 & csr-1 targeting P4Rev TACCGTAGGTGCCCTGTGTA 3317 qChIP his-3 & csr-1 targeting P5Fwd and deletion analysis CCCCTTCCTGCCGTGGGAGA 3562 qChIP his-3 & csr-1 targeting TCAGCAGGCATAGTCAAGACTGGP5Rev and deletion analysis T 4782 qChIP his-3 & csr-1 targeting P6Fwd CCCGCTTCAGCAACCAAGTT 4783 qChIP his-3 & csr-1 targeting P6Rev AACTTTAGCCCGCGTTACGG 3319 qChIP his-3 & csr-1 targeting P7Fwd and deletion analysis TGGGTCGATGGAGTACCTTCCCC 3563 qChIP his-3 & csr-1 targeting TGCACTATCCTTTTCAGGGGCTTP7Rev and deletion analysis GT 3902 qChIP his-3 & csr-1 targeting P8Fwd GACCTACACGGCCCGGGGAA 3903 qChIP his-3 & csr-1 targeting P8Rev ACCGACGAGACTTGACTGCCCA 3986 qChIP his-3 & csr-1 targeting P9Fwd GTGGCGGCGTGAACGGTCAT 3987 qChIP his-3 & csr-1 targeting P9Rev AGTCAAGCCTCGCGATCGTGA 3321 qChIP his-3 & csr-1 targeting P10Fwd TGAACAGGTGACGGCGGGAGT 117 3564 qChIP his-3 & csr-1 targeting P10Rev CGGGTCCGGAGTCCATCACCA 3976 qChIP his-3 & csr-1 targeting P11Fwd AGTGGTCCAGAGTGGGATCGGT 3977 qChIP his-3 & csr-1 targeting P11Rev ACCGCCAATATGGCATCGCCC 3974 qChIP his-3 & csr-1 targeting P12Fwd TGACGGCGCGCAGATTGGAG 3975 qChIP his-3 & csr-1 targeting P12Rev TCGCTTCCCTTCTCCCACCATCC 3978 qChIP his-3 & csr-1 targeting P13Fwd TTGACGGCGCGCAGATTGGAG 3979 qChIP his-3 & csr-1 targeting P13Rev CCACCATCCTTCCCTCTGCCACA 3992 qChIP his-3 & csr-1 targeting P14Fwd CATCGCAGCTCAACCGCAGA 3993 qChIP his-3 & csr-1 targeting P14Rev GCCAGCCGGTGTCAAGACAGA 3904 qChIP his-3 & csr-1 targeting AACAAAGACGCTCTTCTGGTGGCP15Fwd C 3905 qChIP his-3 & csr-1 targeting P15Rev ACTACCAAACTGCCGACGGCT 3943 Construction of Δ47.4 GTAACGCCAGGGTTTTCCCAGTC kb::hph+(N4933) ACGACGTGTGGCTTGCAGGCACGCAA 3944 Construction of Δ47.4 ACCGGGATCCACTTAACGTTACT kb::hph+(N4933) GAAATCAGTCCGAGTGGGCCTGCCTC 3945 Construction of Δ47.4 GCTCCTTCAATATCATCTTCTGTC kb::hph+(N4933) GACGGACCACCACCCAGCGTGGAAAG 3946 Construction of Δ47.4 GCGGATAACAATTTCACACAGGA kb::hph+(N4933) AACAGCTTGCCGCCGGCTGAGAAACC 4960 ALS159 (N5101) qPCR TTGGGATGATTTGGGACGGG 4961 ALS159 (N5101) qPCR TCCCAAGCTGACAGTTCCAC 4961 ALS159 (N5101) qPCR TGCATGCTCTCCCCCTTTTG 4963 ALS159 (N5101) qPCR TCTGAGGGATGTGCCAAACC 4974 NM149 (N5857) qPCR CGCCATTTCTACCCCGATGA 4975 NM149 (N5857) qPCR TGCCAAGCCATCTTTTTGCC 4976 NM149 (N5857) qPCR CTACGGGTTGCTGCCAAGTA 4977 NM149 (N5857) qPCR CCTCAGAGAATCGGGGCATC 4978 NM149 (N5857) qPCR GGGCTCAGTCACTTGCTACA 4979 NM149 (N5857) qPCR GATATACCCGCACCAGCACA 4984 OY337 (N5858) qPCR CTTCGCCTCTCACTCCGATG 118 4985 OY337 (N5858) qPCR GGCAGCTAGCAATCGGTTTT 4986 OY337 (N5858) qPCR GAGCCTGTCCAAGACGACAA 4987 OY337 (N5858) qPCR CGGTGACGGTAGTGTGTAGG 4990 OY337 (N5858) qPCR GTTACTGGCGGGAAATGGGA 4991 OY337 (N5858) qPCR TTGGGACCAGGTTTGTCCAC 4996 OY350 (N5862) qPCR GCCTTGGACCCTCGAATGAA 4997 OY350 (N5862) qPCR TGGGAAAACGTGGGGGAAAA 4998 OY350 (N5862) qPCR TGGGTGAGGTCTTTGGAGGA 4999 OY350 (N5862) qPCR AAGAGTTCCTGAACGTCGCC 5002 OY350 (N5862) qPCR AGGGTTGCTGGTAATCCGTG 5003 OY350 (N5862) qPCR CAAGGCTTGGGGAAAGGGAA 3317 UK3-41 (N5866) qPCR CCCCTTCCTGCCGTGGGAGA 3562 UK3-41 (N5866) qPCR TCAGCAGGCATAGTCAAGACTGGT 3954 UK3-41 (N5866) qPCR GTAGCTAGCGGGTGCTGCCG 3955 UK3-41 (N5866) qPCR AGGCGCCAGGAAGAGTATAGCCC 5012 OY329 (N5102) qPCR AAATCCACTCATCCTCGGCG 5013 OY329 (N5102) qPCR CTCGGATCACCGTCAACAGG 5020 OY329 (N5102) qPCR ATGGTAACGTGGACAGGTGC 5021 OY329 (N5102) qPCR TTGAACGCCGTAGAGGGATG 5028 UK2-32 (N5859) qPCR AGGAAGTACGCCTTGCAGTC 5029 UK2-32 (N5859) qPCR CCTGTATAATGGCGGTCCCC 5034 UK2-32 (N5859) qPCR TCGAACCATGTGAGCTGCTT 5035 UK2-32 (N5859) qPCR GAGAACGCCGAATCGCTCTA 5036 AR16 (N5100) qPCR GAGAACGCCGAATCGCTCTA 5037 AR16 (N5100) qPCR GCCCCCTTTTTGTCGTTAGC 5040 AR16 (N5100) qPCR CGTCAACGGTAGCTGGAAGA 5041 AR16 (N5100) qPCR CCTCTTTGTGTCGAAGCCCA 5042 AR16 (N5100) qPCR TGCCAAAGCACAACAAGCTG 5043 AR16 (N5100) qPCR TTGATACCACGGGCTTCGAC 5046 AR16 (N5100) qPCR GGATCATCGGTAGGTTGGGT 5047 AR16 (N5100) qPCR TCAGATCCAGCTAGTTTCGCC 5316 Telomere repeat FP TTAGGGTTAGGGTTAGGG 5317 Telomere repeat RP CCCTAACCCTAACCC 5318 Targeting tel repeats to csr-1 FP1 GCAATGTCGTCAATGGCTGCAATTAACCCTCACTAAAGGGA 5319 Targeting tel repeats to csr-1 RP1 GTGTGCAGCGCAGGAAATCCATAATACGACTCACTATAGGG 5320 Targeting tel repeats to csr-1 FP2 GTGTGCAGCGCAGGAAATCCAATTAACCCTCACTAAAGGGA 5321 Targeting tel repeats to csr-1 RP2 GCAATGTCGTCAATGGCTGCATAATACGACTCACTATAGGG 5322 Verify LG I circularization FP AGAGGAGTCCGTAGGCGAAT 5323 Verify LG I circularization RP TCGTTCGGTTGACAGCTTGA 119 5324 Verify LG II circularization FP TGTTTCGGCGATGGGAAGAA 5325 Verify LG II circularization RP ACTTCGAGTATGTAGCGGCG 5326 Verify LG III circularization FP CGAGGCTCCATAATGCTCGT 5327 Verify LG III circularization RP TATTATAGGGCGCGCGGAAG 5328 Verify LG IV circularization FP GGCGCAAAAACCTTCCTACC 5329 Verify LG IV circularization RP ACGACAGGGCCTAGGGTAAT 5330 Verify LG VI circularization FP TAGGTTGAAGGCTATCGGCG 5331 Verify LG VI circularization RP CCTTGGTTGCATTTGGTGGG 5332 Verify LG VII circularization FP GCCTTCGGCTACCTTTCCTT 5346 Verify LG VII circularization RP CTCCCTTTCAGCTCGTGTGT 5353 3’ csr-1 qPCR FP CGCCGTTAATGCAGTTGTGAT 5354 3’ csr-1 qPCR RP CCCCAGCAACTGCGTCTATT 5133 NM149 (N5857) breakpoint 1 FP TTGCGGCAAGTTTGAAGTCG 5134 NM149 (N5857) breakpoint 1 RP TGAAGCGTAAGCTCGTGTGT 5130 NM149 (N5857) breakpoint 2 FP GCTCAAAGTGGGGACTGACA 5154 NM149 (N5857) breakpoint 2 RP ATCCTTCTCCGCTGTTTCGG 5123 OY329 (N5102) breakpoint 1 FP GTTGTTGTGGTTTCCTCGCC 5124 OY329 (N5102) breakpoint 1 RP ATATAGGCGTAGCGTTGCCC 5125 OY329 (N5102) breakpoint 2 FP TGTTGCCTGGACTGCTAGTG 5126 OY329 (N5102) breakpoint 2 RP AGCCTAAACCTCGGCTAGGA 5127 OY329 (N5102) breakpoint 3 FP CCGGTATCACGAGCTTCTCC 5128 OY329 (N5102) breakpoint 3 RP GGGCGGAAGTTGAGCTGTAT 5155 UK3-41 (N5866) breakpoint 1 FP GAACGGGACGTTCAAGGCTA 5157 UK3-41 (N5866) breakpoint 1 RP TGCTTGTCTCGTTTTGCAGC 5158 UK3-41 (N5866) breakpoint 2 FP CGGGAGAGGGGGATAGTTGA 5473 UK3-41 (N5866) breakpoint 2 RP CGCACTCACATGCTGCATAC 5156 UK3-41 (N5866) breakpoint 3 FP AAGGCGTAATGGACACGAGG 5472 UK3-41 (N5866) breakpoint 3 RP GCATCGTATTTGCACCGTCC 5476 ALS159 (N5101) breakpoint 1 FP TCCGCAGCCGAAGTTACAAT 5477 ALS159 (N5101) breakpoint 1 RP ACGTCACTCTCTGCCCCTAT 3902 PtrpC-nat1 FP CAACTGATATTGAAGGAGCA 1369 nat-1 RP GGGCATGCTCATGTAGAGCG 3406 TERT KO FP1 AGCGAACTACTACCAACTACG CCCAAAAAATGCTCCTTCAATAT 3407 TERT KO RP2 CAGTTGGCATGTGAGACACTATC ACG CGCTCTACATGAGCATGCCCTGC 3408 TERT KO FP3 CCCTGAAAGTCTGGTACTGGGAT TGG 3409 TERT KO RP4 CTTTCACGACTACCTTCCAAG 6271 6271_NCU02840_qPCR_F CCCTCTCAGACGAGGATATTCA 6272 6272_NCU02840_qPCR_R GCTCTGCTGCTTCTCCTTTAT 6277 6277_NCU04720_qPCR_F TCAGGTTGGAGGAGAGGTATAA 6278 6278_NCU04720_qPCR_R CTATGAGGCCGAAATCCTTGT 6281 6281_NCU10030_qPCR_F CTGCGATCGTAACACTGGATTA 120 6282 6282_NCU10030_qPCR_R CCGTCCGACATGTAATTACTCAG 6283 6283_NCU10031_qPCR_F CCGATGTCCAGAAGCAGTATATTA 6284 6284_NCU10031_qPCR_R CAGAGCAACTGAGTGGATAGTC 3565 Telomere 1L qPCR FP AGCGTTCAAATGCCGTGACCTGT 3566 Telomere 1L qPCR RP AGTCCAATGGTGCTAACGGCGA 5408 5_his-3_FP AAGCTTGCCATCTCCACCATC 5409 5_his-3_RP GGGCGTGCACGGCTATGG 5410 3_his-3_FP GGGCCAAGCTACCCCGTC 5411 3_his-3_RP GATCCAATGCGGATGGATTCGC 5412 T3_his-3_FP1 CCATAGCCGTGCACGCCCATTAACCCTCACTAAAGGGA 5413 T7_his-3_RP1 GACGGGGTAGCTTGGCCCTAATACGACTCACTATAGGG 4776 his-3 qPCR FP AACAGCTGAGGGAGCCAATG 4777 his-3 qPCR RP GTTTCAGGGGTTTCGTTCGC Chapter IV Primers for replacement of NCU04278 with trpC::nat-1 Primer Description Sequence (5’->3’) 6405 NCU04278_5del_FP TTTTCGTGCTTCACACGCTG 6406 NCU04278_5del_RP GCCTCCGCCTCCGCCTCCGCCGCCTCCGCCCG CTGAGGAGGGCCTTCT 6409 NCU04278_3del_FP GAGCTCGGTACCAAGCTTGATGCATAGCCAG AGGGCACAGCTTCGTGG 6410 NCU04278_3del_RP TTGCCTGCGCCAAGATAGAG 4882 nat-1_split_FP GTACAAGTAACAACTGATATTGAAGGAGC 4883 nat-1_split_RP AACCCCATCCGCCGGTACGCG Primers for generation and verification of his-3+::PCCG-1::NCU04278::3xFLAG construct Primer Description Sequence (5’->3’) 6397 NCU04278_his3_FP ACGTACGTTCTAGATGGGCTACTCTGTCTCCT CTTCC (XbaI) 6398 NCU04278_his3_RP ACGTACGTCTTAATTAAACGTGGCGGTCCTG CCG (PacI) 6715 Pccg_seq_FP CAGGCAGCCAGGAACACAAGC 6407 NCU04278_seq1_FP GAACCGGCACCAGTTCCAGA 6426 NCU04278_seq2_FP CTTCACTCAAGCCTAAGACCCTCTC 6427 NCU04278_seq3_FP CGAATGGGGCCGAAGTAACTC 121 RT-qPCR primers Primer Description Sequence (5’->3’) 6271 NCU02840_RTqPCR_FP CCCTCTCAGACGAGGATATTCA 6272 NCU02840_RTqPCR_RP GCTCTGCTGCTTCTCCTTTAT 6687 NCU04991_RTqPCR_FP GCCATTTTGGATCGGTCACAAGTACT 6688 NCU04991_RTqPCR_RP CGACGAAGATAGCCCTGGGA 6581 NCU05173_RTqPCR_FP CGAGTGTGTTGGACCTGACG 6568 NCU05173_RTqPCR_RP CCTGTTCGAGTTATCGGTGTTG 6583 NCU07152_RTqPCR_FP GGTGACCCCAAACCTTATGTCGC 6584 NCU07152_RTqPCR_RP GGCTCGAATCTGCCTCCAGC 6685 NCU09306_RTqPCR_FP GCGGTGATGGAGTATGGCTT 6686 NCU09306_RTqPCR_RP GCAAGTGCTCAGCGCCAT 6595 NCU09604_RTqPCR_FP GGCTAGCCTCCGTCGAGCATG 6596 NCU09604_RTqPCR_RP AATGGCGGGTGCAGATGTAG 6683 NCU09633_RTqPCR_FP ACAGCCTCACCACAACATACCTAATC 6684 NCU09633_RTqPCR_RP CATTGTTTGTCGGGTCTGCG 6589 NCU10038_RTqPCR_FP CCTGGCTGGCACTCGTATGGG 6590 NCU10038_RTqPCR_RP GTCACTGGTGCAGCACTTGG 6689 NCU10070_RTqPCR_FP AGATTCACCGGGTCAAGAGTCATTTATAC 6690 NCU10070_RTqPCR_RP TTGGCTGTTTCTCGCAGGAT ChIP-qPCR primers Primer Description Sequence (5’->3’) 4082 hH4_qPCR_FP CATCAAGGGGTCATTCAC 4083 hH4_qPCR_RP TTTGGAATCACCCTCCAG 5455 Tel_IIIR_qPCR_FP CGGGCTGTTGGTCAGTAGCG 5456 Tel_IIIR_qPCR_RP CAAGCCGCGCCGTCATTACC 6609 NCU07152_qPCR_FP GGCTCTTGGAACCTTACTAGCG 6610 NCU07152_qPCR_RP GGGTCACCTCTTCTGGAAGGC 6611 NCU05173_qPCR_FP GGCGCACACATTGCTCATTTG 6612 NCU05173_qPCR_RP TGGTGGAAAACAGCATCACCC 6707 NCU08251_qPCR_FP TCTGTCCAGCACCAAGTTATC 6708 NCU08251_qPCR_RP TGGCCAGGTACTGCATTAAG 6709 NCU08085_qPCR_FP CAGGAACAGCAGGTGGATATT 6710 NCU08085_qPCR_RP GGACTATTCGAAGGATGGTATGG 6711 NCU05086_qPCR_FP CAAGAGAATGCCAGACCTAGAG 6712 NCU05086_qPCR_RP GGTACTCCAGATGGGAAAGATAG 6713 NCU07801_qPCR_FP ACTGACGTGCCGAGATGAAG 6714 NCU07801_qPCR_RP TTGGGTGACATGATGAGGGC 122 Chapter V Replacement of NCU05173 and NCU07152 ORFs with hph and nat-1 respectively Primer Description Sequence (5’->3’) 6385 NCU07152_5del_FP GAGCAGGTCGTGTCCTCAGG 6386 NCU07152_5del_RP CGTGTCGTCGAGGGTGGCCATCTTGATGGAT TGTCTTGATCGGATGC 6387 NCU07152_3del_FP TGAGCATGCCCTGCCCCTGACGGGTAAATGA CGAGTGTCTGTTGC 6388 NCU07152_3del_RP GGTTCACGAGACAGTCAGCATAGG 6605 NCU05173_5del_FP GCGCTTCTTCAGGCTGAGCG 6606 NCU05173_5del_RP TCGCGGTGAGTTCAGGCTTTTTCATTGTCGCA GTGGTTGATGACTGAGG 6607 NCU05173_3del_FP ACCGGGATCCACTTAACGTTACTGAAATCTC GTGCAGCTGATGACATTGTAACC 6608 NCU05173_3del_RP GGATTGAGAAGAAGTAATAGAAGGCCG 6269 nat-1_ORF_RP TCAGGGGCAGGGCATGCTCATG 6270 nat-1_ORF_FP ATGGCCACCCTCGACGACACG 6625 hph_ORF_FP ATGAAAAAGCCTGAACTCACCGCGA 2895 hph_ORF_RP AGCTGACATCGACACCAACG 2954 hph_internal_FP TCGCCTCGCTCCAGTCAATGACC 2955 hph_internal_RP AAAAAGCCTGAACTCACCGCGACG ChIP-qPCR primer pairs Primer Description Sequence (5’->3’) 3565 Telomere_IL_qPCR_FP AGCGTTCAAATGCCGTGACCTGT 3566 Telomere_IL_qPCR_RP AGTCCAATGGTGCTAACGGCGA 4082 hH4_qPCR_FP CATCAAGGGGTCATTCAC 4083 hH4_qPCR_RP TTTGGAATCACCCTCCAG 6487 NCU08834_qPCR_FP TCCCGATCCTGCTCTAGCTT 6488 NCU08834_qPCR_RP ACTAGTTCGGCTGAGTCCCT 6531 NCU02856_qPCR_FP AGTTTCACACGGGACTGGAC 6532 NCU02856_qPCR_RP TACGTCCACCACTGCTTGTC 6609 NCU07152_5_qPCR_FP GGCTCTTGGAACCTTACTAGCG 6610 NCU07152_5_qPCR_RP GGGTCACCTCTTCTGGAAGGC 6611 NCU05173_5_qPCR_FP GGCGCACACATTGCTCATTTG 6612 NCU05173_5__qPCR_RP TGGTGGAAAACAGCATCACCC 6565 NCU07152_promoter_FP CGGTTCCAAAACTGCCCCTGTG 6645 NCU07152_promoter_RP CTCAGCGGGGTATATCAACGGC 6567 NCU05173_promoter_FP GCATTACCCTCGACAGGGTCTG 6646 NCU05173_promoter_RP GCTACCACCATGTGAAGCTCTGG 6647 BAH_K9_peak_1_FP GAATAAAAAAAGGCTTTTTTATTACTTCC TCGTC 6648 BAH_K9_peak_1_RP GTTCCTATTTTATTTATTTAATTTAAGAG ATTGCGGC 123 RT-qPCR primer pairs Primer Description Sequence (5’->3’) 6271 NCU02840_RTqPCR_FP CCCTCTCAGACGAGGATATTCA 6272 NCU02840_RTqPCR_RP GCTCTGCTGCTTCTCCTTTAT 3209 NCU04173_Actin_RTqPCR_FP AATGGGTCGGGTATGTGCAA 3210 NCU04173_Actin_RTqPCR_RP CTTCTGGCCCATACCGATCAT 6581 NCU05173_RTqPCR_FP CGAGTGTGTTGGACCTGACG 6568 NCU05173_RTqPCR_RP CCTGTTCGAGTTATCGGTGTTG 6583 NCU07152_RTqPCR_FP GGTGACCCCAAACCTTATGTCGC 6584 NCU07152_RTqPCR_RP GGCTCGAATCTGCCTCCAGC 6599 NCU07241_RTqPCR_FP CTGATACCGACATCTCCGTAACAG 6600 NCU07241_RTqPCR_RP CCAATCATGGGACCAGCCCAAG 6593 NCU07246_RTqPCR_FP GATACCAGGGCACCTGGATC 6534 NCU07246_RTqPCR_RP GGCGTTCTTTGCCGACTTAC 6615 NCU07624_RTqPCR_FP CCCAGGGGCGACAAGCAACC 6616 NCU07624_RTqPCR_RP CAGAAATCATGTCAGCGCGTATGC 6591 NCU08097_RTqPCR_FP GTTACGCAAGGGGCAACACCTAC 6592 NCU08097_RTqPCR_RP GAGCTGCGGTATCATCGGTG 6585 NCU08570_RTqPCR_FP CCACGAAGCGCCAACATGACG 6586 NCU08570_RTqPCR_RP GATGTCGTGATGTGGCTCCACG 6587 NCU09178_RTqPCR_FP GCTCGTATCATCCTTACGACATGG 6588 NCU09178_RTqPCR_RP CTAGAACTCTCATCATCGCTCCC 6603 NCU09274_RTqPCR_FP CCTATTATGCTTGGACCACAACG 6604 NCU09274_RTqPCR_RP ACATTTGGATGCGTCTGCCC 6595 NCU09604_RTqPCR_FP GGCTAGCCTCCGTCGAGCATG 6596 NCU09604_RTqPCR_RP AATGGCGGGTGCAGATGTAG 6613 NCU09640_RTqPCR_FP CTCGTCTTTTATCTTGCACTTTACTT CC 6614 NCU09640_RTqPCR_RP GCCAAAATGTGGTGATGAGCC 6597 NCU09990_RTqPCR_FP GCAAGGATAGCAAAGCATGGGAGG 6598 NCU09990_RTqPCR_RP CCCGTTGTTGCAATTCTTCCAATCG 6589 NCU10038_RTqPCR_FP CCTGGCTGGCACTCGTATGGG 6590 NCU10038_RTqPCR_RP GTCACTGGTGCAGCACTTGG 6601 NCU16720_RTqPCR_FP GGAGGTTTGGCAGTTCACCAAGG 6602 NCU16720_RTqPCR_RP GCGGATGACTGGACGCTCTCC Southern Probes Primer Description Sequence (5’->3’) 1665 his-3_FP GACGGGGTAGCTTGGCCCTAATTAACC 3128 his-3_RP CGATTTAGGTGACACTATAG 5271 Tel_VIIL_FP GGCATCCGTGGGTGTCCCAG 5272 Tel_VIIL_RP TTCCCGTCCCTACCAGGCAT 6567 NCU05173_FP GCATTACCCTCGACAGGGTCTG 6568 NCU05173_RP CCTGTTCGAGTTATCGGTGTTG 124 DamID-seq protocol Primer Description Sequence (5’->3’) 5048 AdR-top CTAATAACGACTCACTATAGGGCAGCGTGG TCGCGGCCGAGGA 5049 AdR-bottom TCCTCGGCCG 5050 AdR-top + AdR-bottom (see above) (annealed) 5051 Bio-AdR Biotin-GGTCGCGGCCGAGGATC Cloning the BAH domain of EPR-1 Primer Description Sequence (5’->3’) 6681 BAH domain F AGATCCGGATCCTTAGCATAAAGAAAAAGGACCC AAAACAAGG 6682 BAH domain R AGATCCGCGGCCGCTTAGGTCGAGAGTTCTTGAGA CCG Generation and verification of his-3+::pCCG::N-GFP::EPR-1 constructs Primer Description Sequence (5’->3’) 6416 NCU07505_his- ACGTACGTTTAATTAACGCCTCCTCGCG 3_Ntag_FP CAAGAGA (PacI) 6417 NCU07505_his- ACGTACGTCTCTAGATGCTCACTCGCTT 3_Ntag_RP GCATCAT (XbaI) 6367 NCU07505_W184A_FP TCTTCGCCAGAGTTTACGCGATGTACTG GCCTGACG 6368 NCU07505_W184A_RP CGTCAGGCCAGTACATCGCGTAAACTCT GGCGAAGA 6399 NCU07505_W292A_FP CTGCTCTTCAGAATCATGTAAGAAAGCG TTACACGAAGAATGCATCAAGGACC 6400 NCU07505_W292A_RP GGTCCTTGATGCATTCTTCGTGTAACGCT TTCTTACATGATTCTGAAGAGCAG 6425 N-GFP_seq_FP TCCTGCTGGAGTTCGTGACC 6346 NCU07505_seq_FP1 TGGACCGACATGACGAGGTA 6348 NCU07505_seq_FP2 GGAAAGCAAAGCCAAGACGG 6366 NCU07505_seq_FP3 GACGAGGAGACCCAGGATAGTC Replacement of epr-1 with trpC::nat-1 Primer Description Sequence (5’->3’) 6401 NCU07505_5del_FP TGGCTGAGTGCAACGATTCT 6402 NCU07505_5del_RP GCCTCCGCCTCCGCCTCCGCCGCCTCCGCCTG TTGAGTAGTGTGAGTGTAGTAGAAGAG 6350 NCU07505_3_FP GAGCTCGGTACCAAGCTTGATGCATAGCTAG TGATGGCGGACGATTACGG 125 6351 NCU07505_3_RP TGCTCACTCGCTTGCATCAT 4882 nat-1_split_FP GTACAAGTAACAACTGATATTGAAGGAGC 4883 nat-1_split_RP AACCCCATCCGCCGGTACGCG Endogenous C-terminal tagging of EPR-1 with 10xGly::Dam (WT and PHD) Primer Description Sequence (5’->3’) 6348 NCU07505_5_FP GGAAAGCAAAGCCAAGACGG 6349 NCU07505_5_RP GCCTCCGCCTCCGCCTCCGCCGCCTCCGCC ATTAACCAACACACCACACACCAAA 6350 NCU07505_3_FP GAGCTCGGTACCAAGCTTGATGCATAGCT AGTGATGGCGGACGATTACGG 6351 NCU07505_3_RP TGCTCACTCGCTTGCATCAT 6346 NCU07505_5alt_FP TGGACCGACATGACGAGGTA 6399 NCU07505_W292A_FP CTGCTCTTCAGAATCATGTAAGAAAGCGTT ACACGAAGAATGCATCAAGGACC 6400 NCU07505_W292A_RP GGTCCTTGATGCATTCTTCGTGTAACGCTT TCTTACATGATTCTGAAGAGCAG 4882 nat-1_split_FP GTACAAGTAACAACTGATATTGAAGGAGC 4883 nat-1_split_RP AACCCCATCCGCCGGTACGCG 126 APPENDIX D PLASMID TABLES Chapter II Plasmid Description 3130 pZero::3xFLAG::nat-1::loxP 3167 histone H3 codon optimized 3168 histone H3 codon optimized (H3K56R) 3169 histone H3 codon optimized (H3K56Q) Chapter III Plasmid Description 1991 pBM61 – his-3 targeting vector 3110 LG VIL H3K27me2/3 segment #1 cloned into 1991 3111 LG VIL H3K27me2/3 segment #2 cloned into 1991 3112 LG VIL H3K27me2/3 segment #3 cloned into 1991 3113 LG VIL H3K27me2/3 segment #4 cloned into 1991 3114 LG VIL H3K27me2/3 segment #5 cloned into 1991 3115 LG VIL H3K27me2/3 segment #6 cloned into 1991 3116 LG VIL H3K27me2/3 segment #7 cloned into 1991 3117 LG VIL H3K27me2/3 segment #8 cloned into 1991 3172 (TTAGGG)76 cloned into pCR4-TOPO-TA FGSC #10598 pAL12-Lifeact – source of trpC::nat-1 Chapter IV Plasmid Description 2401 PCCG-1::C-Gly::3xFLAG 3237 (FGSC 10598) pAL12 – source of nat-1 3340 PCCG-1::NCU04278WT::C-Gly::3xFLAG Chapter V Plasmid Description 1991 pBM61 – his-3+ targeting vector 2406 his-3+::pCCG::N-GFP 3329 his-3+::pCCG::N-GFP::NCU07505 3345 his-3+::pCCG::N-GFP::NCU07505W184A 3346 his-3+::pCCG::N-GFP::NCU07505W292A 127 3130 pZero::10xGly::3xFLAG::trpC::nat-1 3131 pZero::10xGly::Dam::trpC::nat-1 3237 (FGSC 10598) pAL12 – source of nat-1 2283 pCSN44 – source of hph 3230 pE-SUMO 3410 pE-SUMO + BAHEPR-1 128 REFERENCES CITED 1. 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