cohesin impedes heterochromatin assembly in fission yeast ... · an acetylation-mimicking mutation...

HIGHLIGHTED ARTICLE| INVESTIGATION

Cohesin Impedes Heterochromatin Assembly in FissionYeast Cells Lacking Pds5

H. Diego Folco, Andrea McCue,1 Vanivilasini Balachandran, and Shiv I. S. Grewal2

Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda,Maryland 20892

ABSTRACT The fission yeast Schizosaccharomyces pombe is a powerful genetic model system for uncovering fundamental principlesof heterochromatin assembly and epigenetic inheritance of chromatin states. Heterochromatin defined by histone H3 lysine 9 meth-ylation and HP1 proteins coats large chromosomal domains at centromeres, telomeres, and the mating-type (mat) locus. Althoughgenetic and biochemical studies have provided valuable insights into heterochromatin assembly, many key mechanistic details remainunclear. Here, we use a sensitized reporter system at themat locus to screen for factors affecting heterochromatic silencing. In additionto known components of heterochromatin assembly pathways, our screen identified eight new factors including the cohesin-associatedprotein Pds5. We find that Pds5 enriched throughout heterochromatin domains is required for proper maintenance of heterochromatin.This function of Pds5 requires its associated Eso1 acetyltransferase, which is implicated in the acetylation of cohesin. Indeed, introducingan acetylation-mimicking mutation in a cohesin subunit suppresses defects in heterochromatin assembly in pds5D and eso1D cells. Ourresults show that in cells lacking Pds5, cohesin interferes with heterochromatin assembly. Supporting this, eliminating cohesin from themat locus in the pds5D mutant restores both heterochromatin assembly and gene silencing. These analyses highlight an unexpectedrequirement for Pds5 in ensuring proper coordination between cohesin and heterochromatin factors to effectively maintain genesilencing.

KEYWORDS heterochromatin; fission yeast; mat locus; Pds5; cohesin acetylation

THE eukaryotic genome is regulated by epigenetic modifi-cations that create structurally distinct “open” euchroma-tin and “closed” heterochromatin domains. Heterochromatinis defined by hypoacetylation of histones and methylation ofhistone H3 at lysine 9 (H3K9me), and is implicated in diversefunctions including transcriptional and post-transcriptionalsilencing, as well as the maintenance of genome stability(Grewal and Jia 2007; Wang et al. 2016; Freitag 2017;Allshire andMadhani 2018). Understanding the mechanismsof heterochromatin assembly is vital to elucidate the causesof human diseases linked to defects in this process.

The fission yeast Schizosaccharomyces pombe is an idealmodel genetic organism for studying heterochromatin assem-bly pathways. The S. pombe genome contains facultative het-erochromatin islands as well as constitutive heterochromatindomains coating centromeres, telomeres, and the silentmating-type (mat) region (Cam et al. 2005). At centromeres, arrays ofrepetitive dg and dh elements embedded within pericentro-meric regions serve as heterochromatin nucleation centers,whereas at the mat locus a cenH element bearing homologyto dg and dh repeats serves to nucleate heterochromatin(Grewal and Jia 2007). The hallmark heterochromatin mod-ification H3K9me is added by the sole methyltransferase Clr4(SUV39H in mammals) (Nakayama et al. 2001b), which ex-ists in the multisubunit H3K9 methyltransferase (CLRC) pro-tein complex (Hong et al. 2005; Horn et al. 2005; Jia et al.2005). Once added, H3K9me spreads across extended chro-mosomal domains surrounded by boundary DNA elements(Hall et al. 2002). Spreading requires a unique feature ofClr4 to both “read” and “write” H3K9me (Nakayama et al.2001b; Zhang et al. 2008; Al-Sady et al. 2013). Clr4-mediatedH3K9me deposition also provides binding sites for the

Copyright © 2019 by the Genetics Society of Americadoi: https://doi.org/10.1534/genetics.119.302256Manuscript received April 26, 2019; accepted for publication June 24, 2019; publishedEarly Online July 5, 2019.Supplemental material available at Figshare: https://doi.org/10.25386/genetics.8307248.1Present address: Department of Molecular Genetics, The Ohio State University,Columbus, OH 43210.

2Corresponding author: Laboratory of Biochemistry and Molecular Biology, NationalCancer Institute, National Institutes of Health, Bethesda, MD 20892. E-mail: [email protected]

Genetics, Vol. 213, 127–141 September 2019 127

https://doi.org/10.1534/genetics.119.302256https://doi.org/10.25386/genetics.8307248https://doi.org/10.25386/genetics.8307248mailto:[email protected]:[email protected]

chromodomains of HP1 (Heterochromatin Protein 1) familyproteins such as Chp2 and Swi6 (Thon and Verhein-Hansen2000; Bannister et al. 2001; Nakayama et al. 2001b; Sadaieet al. 2004), which in turn create a platform for the recruit-ment of effectors such as SHREC [Snf2-histone deacetylase(HDAC) repressor complex] containing the Clr1, Clr2, Clr3,and Mit1 proteins (Thon and Klar 1992; Ekwall and Ruusala1994; Thon et al. 1994; Sugiyama et al. 2007; Job et al.2016). Interestingly, Swi6 also recruits the cohesin-loadingcomplex that is critical for preferential cohesin enrichment atheterochromatic loci (Bernard et al. 2001; Nonaka et al.2002; Fischer et al. 2009).

Paradoxically, heterochromatin nucleation is dependentupon transcription by RNA polymerase II. Highly conservedRNA-processing factors, including RNA interference (RNAi)-dependent and -independent mechanisms, target coding andlong noncodingRNAs to assemble facultative and constitutiveheterochromatin (Reyes-Turcu and Grewal 2012; Allshireand Madhani 2018). Transcripts produced from repeat ele-ments at constitutive heterochromatin domains are pro-cessed by RNAi machinery into small interfering RNAs(siRNAs). The siRNAs bind to Argonaute (Ago1) and providespecificity for targeting the RNAi-induced transcriptionalsilencing complex, which facilitates recruitment of CLRC(Verdel et al. 2004; Zhang et al. 2008; Bayne et al. 2010).RNAi-independent mechanisms involving RNA-processingand RNA polymerase II termination factors operate in paral-lel to RNAi to mediate the assembly of constitutive hetero-chromatin domains (Reyes-Turcu et al. 2011; Marina et al.2013; Chalamcharla et al. 2015; Tucker et al. 2016; Touat-Todeschini et al. 2017).

Early studies of the mat locus in fission yeast were partic-ularly valuable in uncovering mechanisms directing the nu-cleation, spreading, and propagation of heterochromatinthrough mitosis and meiosis (Hall et al. 2002; Klar 2007).The mating-type region is comprised of three cassettes:mat1,mat2, andmat3 (Figure 1A). In wild-type (WT) homo-thallic cells (h90), the mating type is determined by the allelepresent in the activemat1 cassette, which is eithermat1P in P(plus) or mat1M in M (minus) cells (Klar 2007). Cells switchmating type by copying genetic information from silentmat2ormat3 donor cassettes that harbor the P andM information,respectively. Silencing at donor mating-type cassettes isenforced by two mechanisms. First, heterochromatin nucle-ated at the cenH element, which is located betweenmat2 andmat3, spreads across a �20-kb region surrounded by bound-ary elements (referred to as IR-L and IR-R) to repress geneexpression across the entire domain (Grewal and Klar 1997;Noma et al. 2001; Hall et al. 2002). Second, the cis-actingelements REII and REIII, which are located adjacent to mat2andmat3 cassettes, respectively (Thon et al. 1994, 1999), actlocally to recruit HDACs and silence donor cassettes indepen-dently of heterochromatin (Grewal et al. 1998; Cam et al.2008). The redundancy in silencing mechanisms underscoresthe fundamental importance of silencingmat2 andmat3 loci.Indeed, impairment of both silencing pathways results in

coexpression of M and P mating-type alleles in haploid cells,leading to an aberrant sporulation phenotype called “haploidmeiosis” (Thon et al. 1994).

Despite significant progress, it is not fully understood howheterochromatic structures are propagated in the context ofothernuclearprocesses. Togain insight into thesemechanisms,we performed a systematic screen for additional factors re-quired for heterochromatic silencing at the silent mat region.Our screen identified several factors with previously unde-scribed roles in heterochromatin regulation. Among the geneswe identified was Pds5, a conserved protein known to asso-ciate with cohesin. Our results suggest that defective hetero-chromatin assembly in cells lacking Pds5 is functionallyconnected to its role in promoting the acetylation of cohesinby the Eso1 acetyltransferase. Our discovery reveals an unex-pected and intriguing role for Pds5 and Eso1 in preventingdeleterious effects of cohesin on heterochromatin assembly.

Materials and Methods

Yeast strains and deletion library

Standard procedures were used for fission yeast cell cultureand genetic manipulations (Sabatinos and Forsburg 2010).S. pombe strains used in this study are listed in SupplementalMaterial, Table S1. Gene deletions and epitope tagging wereperformed by homologous recombination (Bähler et al.1998). The following mutant alleles and reporters were pre-viously described: KD::ade6+ (Ayoub et al. 1999), KD::ura4+

(Grewal and Klar 1996), Kint2::ura4+ (Grewal and Klar1997), REIID mat2P::ura4+ (Thon et al. 1994), psc3-4T(Nonaka et al. 2002), psm3KK/QQ (Kagami et al. 2011),rad21-K1 (Tatebayashi et al. 1998), and rpl42(sP56Q )(Roguev et al. 2007). The haploid deletion library version4.0 (Bioneer, Daejeon, Korea), which contains 3400 strains,was used to systematically screen for factors involved in het-erochromatin formation. We obtained data for 3042 strains,which represent �90% of all nonessential genes in fissionyeast. The tester strain SPDF734 was created as follows.The his2 gene was deleted with a nourseothricin resistancecassette (NAT) in a heterothallic strain (mat1M-smt0) har-boring the REII deletion and a ura4+ marker insertion(mat2P::ura4+). The mat locus can be followed with theNAT marker given its genetic linkage with his2 (, 0.5 cM).To prevent diploids from forming in the first round of screen-ing, the strain also included the rpl42(sP56Q) allele, whichconfers resistance to cycloheximide.

Genetic screen

All manipulations were performed with a multiblot replicatorin a 96-well plate format (Figure S1A) (V&P Scientific, SanDiego, CA). The entire deletion library, consisting of 36plates, was crossed to SPDF734 in SPAS (sporulation agarwith supplements) medium for$ 3 days at 26�. Spores weregerminated in YES (yeast extract plus supplements) plus cy-cloheximide (100 mg/ml), ClonNat (100 mg/ml), and G418

128 H. D. Folco et al.

(100 mg/ml) for $ 3 days at 32�. The multiple selectionstrategy ensured that haploid progeny harbored each deletedgene in the sensitizedmat locus background. Next, cells werereplicated onto PMG5S (pombe minimal glutamate plus fivesupplements) plus cycloheximide (200 mg/ml), ClonNat(200 mg/ml), and G418 (300 mg/ml) and incubated for2 days at 26�, and then exposed to iodine vapor for the initialidentification of candidates. Candidates were then examinedfor haploid meiosis using a standard bright-field microscope.False positives displaying azygotic (from diploids) or zygoticasci (from mating) were eliminated. The verified positivecandidates (Table S2) were tested for haploid meiosis (i.e.,iodine staining) and expression of mat2P::ura4+ in plate di-lution assays employing a combined system that scores fourtraits as follows: (a) PMG5S: iodine staining as 1 = black,0.5 = brown/variegated, and 0 = yellow; (b) PMG – URA:iodine staining [same as for (a)]; (c) PMG + 5-flourooroticacid (FOA): growth as 1= none, 0.5 =medium, and 0= full;and (d) PMG – URA: growth as 1 = full, 0.5 = medium, and

0 = none (Table S3). Only candidates with a score$ 2 werecarried forward for further analyses. Strains carrying dele-tions in the genes of interest were genotyped by PCR, andsubjected to tetrad dissection and random spore analyses toconfirm the phenotypes. Finally, cells from the shortlistedcandidates were spotted on PMG5S for 3 days at 30�. Thepercentage of cells undergoing haploid meiosis was quanti-fied using a bright-field microscope and candidates thatexhibited , 2% haploid meiosis were discarded.

Oligonucleotides, RT-PCR, and quantitative PCR

Oligonucleotides used for chromatin immunoprecipitation(ChIP)-quantitative PCR (qPCR) and RT-qPCR are listed inTable S4. RNAwas isolated using the MasterPure Yeast RNAPurification Kit (Epicentre) according to the manufacturer’sprotocol and random primers were used for cDNA amplifica-tion. qPCR reactions were performed in a Quant Studio 3Real-Time PCR system (Thermo Fisher Scientific) usingSYBR Green Supermix (Bio-Rad, Hercules, CA).

Figure 1 Genetic screen for new factors in-volved in heterochromatin formation. (A)Schematic representation of the mating-type(mat) region in fission yeast. (B) Depiction ofthe genetic screen used to identify new het-erochromatin factors. The screen exploitsthe haploid meiosis phenomenon to identifyfactors required to maintain silencing at themat locus. A tester strain containing the REIIdeletion and mat2P::ura4+ reporter wascrossed to the fission yeast gene deletionlibrary in a 96-well format. Haploid mutantcolonies carrying a silencing reporter werethen screened for derepression of the matlocus, which leads to the expression of bothmating-type factors and haploid meiosis, byiodine staining. See details in the Materialsand Methods section. (C) Results from thescreen indicating the number of candidategenes remaining after each validation step.(D) Comparison of the iodine staining resultsobtained for strains carrying deletions of thenewly identified factors. The percentage ofcells undergoing haploid meiosis is indicatedbelow. N. 200. (E) Analysis ofmat2P::ura4+

expression in strains carrying deletions of thenewly identified factors. Ten-fold serial dilu-tions of the indicated strains were spottedon YEA rich media, with or without the ad-dition of FOA and grown at 32�. WT andclr4D strains serve as growth controls. FOA,5-flouroorotic acid; KAN, kanamycin; NAT,nourseothricin; WT, wild-type; YEA, yeast ex-tract plus adenine.

Effect of Pds5 on Heterochromatin 129

Chromatin immunoprecipitation

ChIP and ChIP-chip experiments were performed as previouslydescribed (Cam et al. 2005; Zofall et al. 2016). Anti-HA (12CA5,Roche or 16B12; BioLegend), anti-GFP (ab290; Abcam), anti-H3K9me2 (ab1220 and ab115159; Abcam), and anti-H3K9me3(ab8898) antibodies were used for immunoprecipitation. DNAisolated from immunoprecipitated chromatin or fromwhole-cellextracts was labeled with Cy5/Cy3 for microarray-based ChIP-chip analyses using a custom 4 3 44K oligonucleotide array(Agilent). Cy5/Cy3 ratios were further processed by a seven-probe sliding window filter to reduce noise.

Data availability

Strains are available upon request. The authors affirm that alldata necessary for confirming the conclusions of the article arepresentwithin the article, figures, and tables.Microarray dataare available at the National Center for Biotechnology In-formation Gene Expression Omnibus repository under theaccession number GSE130233. Supplemental material avail-able at Figshare: https://doi.org/10.25386/genetics.8307248

Results

Sensitized genetic screen for heterochromaticsilencing factors

Genetic screens have proven valuable in identifying factorsinvolved in heterochromatin assembly (Thon and Klar 1992;Ekwall and Ruusala 1994; Thon et al. 1994; Bayne et al. 2014;Zofall et al. 2016; Taneja et al. 2017; Jahn et al. 2018). Touncover additional factors, we used a sensitized reporter sys-tem to screen the S. pombe gene deletion library for factorsaffecting silencing at the silent mat region. We employed anonswitchable (mat1-Msmt0) tester strain carrying a deletionof the local silencerREII, which locally recruits HDACs (Grewalet al. 1998; Cam et al. 2008) near themat2P cassette (Figure 1,A and B) (Thon et al. 1994). In this strain, mat2P silencing isentirely dependent upon heterochromatin formation acrossthe silent mat region. Defects in heterochromatin lead to lossof mat2P silencing, which triggers haploid meiosis resultingfrom coexpression ofM and P information in otherwise haploidcells. Colonies formed bymutants defective inmat2P silencingstain dark brown/black in the presence of iodine vapor due tothe starch-like compound produced by cells undergoing hap-loid meiosis. In contrast, colonies formed byWT cells that lackthis compound stain yellow. A ura4+marker adjacent tomat2P(mat2P::ura4+) provided an orthogonal readout for hetero-chromatic silencing, as evidenced by growth on counterselec-tive medium containing FOA.

To study the effects of individual gene deletions on het-erochromatic silencing, we crossed the tester strain to theS. pombe deletion library (Figure 1B and Figure S1A). Sincethe mat reporter in our tester strain is marked with a tightlylinked NAT marker, and gene deletions in the library wereconstructed using a kanamycin (KAN) marker, we used selec-tion on NAT and G418 plates to obtain the desired segregants.

In addition, the recessive cycloheximide resistance gene (theP56Q allele of the ribosomal protein gene rpl42+) allowedpositive selection for haploid cells of interest while eliminatingdiploids bearing the dominant cycloheximide-sensitive gene(Roguev et al. 2007). Of the deletion mutants, . 100 testedpositive for iodine staining (Figure 1C). The haploid meiosisphenotype of these candidates was further examined by mi-croscopy. Fifty-two candidates that scored positive were se-lected for further analyses (Table S2). We then scored thedegree of haploidmeiosis and themat2P::ura4+ silencing phe-notype, based on the intensity of iodine staining and growth ofmutants on medium either lacking uracil or containing FOA.A maximum score of 4 was assigned to mutants that showedthe highest derepression in both assays (Table S3 andMaterials and Methods). Candidates with scores, 2 were dis-carded. This scoring strategy resulted in a final list of 26 genesthat are required for silencing at the mat region (Table 1).

Identification of new heterochromatic silencing factors

Our screen revealed 18 previously reported factors alongwitheight factors with no described role in heterochromatic si-lencing (Figure 1C and Table 1). As expected, the majorityof strains carrying deletions in known heterochromatin as-sembly factors exhibited the highest scores for haploid mei-osis and derepression of mat2P::ura4+ (Table S3). Theseincluded components of CLRC (Horn et al. 2005), HP1 familyproteins Swi6 and Chp2 (Thon and Verhein-Hansen 2000;Sadaie et al. 2004), components of SHREC (Sugiyama et al.2007; Job et al. 2016), and chromatin remodelers and his-tone chaperones such as Fft3, Hip1, and Ani1 (Blackwell et al.2004; Strålfors et al. 2011; Yamane et al. 2011; Taneja et al.2017; Jahn et al. 2018; Tan et al. 2018). The new genesidentified encoded unnamed products, ribosomal proteins,Yap18, the proteasome regulatory subunit Rpn10 (Wilkinsonet al. 2000), and the cohesin-associated protein Pds5(Hartman et al. 2000; Panizza et al. 2000; Sumara et al.2000; Tanaka et al. 2001; Wang et al. 2002). We namedthe unannotated SPCP1E11.10 and SPAC17C9.15c genesdhm1 and dhm2 (deleterious haploid meiosis), respectively.dhm1 encodes an�23-kDaprotein containing ankyrin repeats,whereas dhm2 encodes an S. pombe-specific small protein(�11 kDa) that lacks annotated domains. Both of these pro-teins exhibit a nuclear localization (Matsuyama et al. 2006).

We independently generateddeletionsof eachof thenewlyidentified genes. The deletion mutants showed haploid mei-osis that closely correlatedwithderepressionofmat2P::ura4+

(Figure 1, D and E). In particular, cells carrying a deletion ofpds5 displayed a high level of haploid meiosis and alleviationof mat2P::ura4+ reporter gene silencing. Since Pds5 is asso-ciated with the cohesin protein complex (Tanaka et al. 2001;Wang et al. 2002; Schmidt et al. 2009), which is enriched atheterochromatic loci (Bernard et al. 2001; Nonaka et al.2002; Fischer et al. 2009), we focused our further effortson understanding the possible function(s) of this conservedprotein in heterochromatin assembly.


https://doi.org/10.25386/genetics.8307248

Loss of Pds5 causes variegated expression and defectiveheterochromatin assembly

Interestingly, in contrast to the uniform staining patterns of WT(yellow) and clr4D cells (black), pds5D cells formed coloniesthat were differentially stained to yield a mixture of yellow andblack patches (Figure 2A). A similar phenotype was also dis-played by several other mutants identified in our screen (FigureS1B and Table S2). This variegated staining pattern is a char-acteristic of mutants that are known to be defective in themain-tenance of heterochromatin and that show a reduction, but notloss, of H3K9me levels (Taneja et al. 2017). Indeed, ChIP anal-yses of H3K9 di- and trimethylation (H3K9me2/3) showed areduction in heterochromatic H3K9 marks at or near mat2P inpds5D (Figure 2B). The decrease correlated with higher expres-sion levels of mat2-Pc and the ura4+ reporter compared to WT(Figure 2C). Thus, the loss of silencing and triggering of haploidmeiosis that are observed in cells lacking Pds5 are functionallyconnected to a failure in the proper assembly of heterochroma-tin at the mat locus.

Pds5 is broadly required for heterochromatin assembly

We next wondered if Pds5 affects H3K9me at other hetero-chromatin domains. Genome-wide analyses of WT and pds5D

cells revealed that H3K9me2 was notably decreased at cen-tromeres and telomeres in pds5D (Figure 3, A and B). Thisresult is consistent with ChIP-qPCR analysis showing a re-duction, but not a complete loss, of both H3K9me2 andH3K9me3 at centromeric repeat elements (Figure 3C). To-gether, our results suggest that the function of Pds5 in het-erochromatin assembly is not limited to themat locus, and infact that Pds5 plays a broader role in heterochromatin forma-tion at other loci.

Pds5 is required for heterochromatin maintenance

Ourpreviousstudieshaveshownthatheterochromatincanself-propagate via an epigenetic mechanism, wherein parentalH3K9 methylated nucleosomes recruit Clr4 to stably maintainthe heterochromatic state in cis both during mitosis and mei-osis (Grewal and Klar 1996; Nakayama et al. 2000; Hall et al.2002; Zhang et al. 2008). The crucial requirement for theepigenetic propagation of heterochromatin is unveiled in theabsence of de novo nucleation mechanisms. Indeed, the reten-tion of parental methylated nucleosomes, and the ability ofClr4 to read and write H3K9me, are essential for clonal prop-agation of heterochromatin in cells lacking the cenH nucleationsite at the mat locus (Zhang et al. 2008; Aygün et al. 2013).

Table 1 List of genes identified in this study

Gene ID Name Description Synonyms Human ortholog

Chromatin remodelers & chaperonesSPBC1347.02 ani1 CENP-A N-terminus domain isomerase Ani1 fkbp39SPAC25A8.01c fft3 SMARCAD1 family ATP-dependent DNA helicase Fft3 snf2SR SMARCAD1SPBC31F10.13c hip1 Histone H3.3 H4 chaperone, hira family Hip1 hir1 HIRASPBC609.05 pob3 Histone H2A-H2B chaperone, FACT complex subunit Pob3 SSRP1SPBC15D4.03 slm9 Histone H3.3 H4 chaperone, hira family Slm9 HIRA

Heterochromatin proteinsSPBC16C6.10 chp2 Heterochromatin (HP1) family chromodomain protein Chp2 CBX1, CBX3, CBX5SPAC664.01c swi6 Heterochromatin (HP1) family chromodomain protein Swi6 SPAC824.10c CBX1, CBX3, CBX5

Histone deacetylasesSPBC2D10.17 clr1 SHREC complex intermodule linker subunit Clr1SPAC1B3.17 clr2 Chromatin silencing protein Clr2SPBC800.03 clr3 Histone deacetylase (class II) Clr3 HDAC6, HDAC10SPBP35G2.10 mit1 SHREC complex ATP-dependent DNA helicase subunit Mit1 CHD3

H3K9 methyltransferase complexSPBC428.08c clr4 Histone H3 methyltransferase Clr4 SUV39H1, SUV39H2SPCC613.12c raf1 CLRC ubiquitin ligase complex WD repeat subunit Raf1/Dos1 dos1, cmc1, clr8SPCC970.07c raf2 CLRC ubiquitin ligase complex subunit Raf2 dos2, cmc2, clr7SPCC11E10.08 rik1 CLRC ubiquitin ligase complex WD repeat protein Rik1 DDB1

OthersSPBC428.07 meu6 Pleckstrin homology domain protein Meu6SPAC694.06c mrc1 Claspin, Mrc1 CLSPNSPAC110.02 pds5 Mitotic and meiotic cohesin loader subunit Pds5 PDS5A, PDS5BSPCC338.16 pof3 F-box protein Pof3 STIP1SPAC637.10c rpn10 19S proteasome regulatory subunit Rpn10 pus1 PSMD4SPBC19F8.03c yap18 ENTH/VHS domain protein (predicted) SNAP91, PICALM

Ribosomal proteinsSPCC663.04 rpl39 60S ribosomal protein L39 RPL39SPBC11C11.09c rpl502 60S ribosomal protein L5 rpl5-2, rpl5b RPL5SPAC959.08 rpl2102 60S ribosomal protein L21 (predicted) rpl21-2, rpl21 RPL21

UnannotatedSPCP1E11.10 dhm1 Ankyrin repeat protein, unknown biological roleSPAC17C9.15c dhm2 Schizosaccharomyces-specific protein

Newly identified factors required for heterochromatic silencing are highlighted in bold. CLRC, multisubunit H3K9 methyltransferase protein complex; ENTH, epsin N-terminalhomology; FACT, facilitates transcription activation; ID, identifier; SHREC, Snf2-histone deacetylase repressor complex; VHS, Vps27p, Hrs and STAM;WD, tryptophan-aspartic acid.


The variegated expression pattern of pds5D was charac-teristic of mutants defective in epigenetic inheritance ofheterochromatin (Figure 2A) (Taneja et al. 2017). To de-termine if Pds5 affects epigenetic stability, we testedwhether the heterochromatic “OFF” state in cells carryinga replacement of cenHwith ade6+ (KD::ade6+) (Ayoub et al.1999) could be maintained in the absence of Pds5. Since theepigenetically maintained OFF state is stably propagatedthrough meiosis (Grewal and Klar 1996; Ayoub et al.1999), we introduced pds5D by a genetic cross. Tetrad anal-yses of meiotic segregants showed that the KD::ade6+ OFFstate was maintained in WT progeny, as expected (Figure 4A).In contrast, maintenance of KD::ade6+ was severely af-fected in pds5D segregants, as indicated by growth on me-dium lacking adenine (Figure 4A). Loss of KD::ade6+

silencing in the pds5D background was confirmed by replicaplating onto low-adenine medium plates. All KD::ade6+

pds5D segregants formed white colonies indicating loss ofade6+ silencing (Figure 4A). Defective silencing correlatedwith a reduction in H3K9me3 levels at the mat region (Fig-ure 4B). A similar loss of silencing occurred in pds5D cellscarrying a KD::ura4+ reporter (Figure S2A), which also cor-related with reduced levels of H3K9me3 (Figure S2B). Im-portantly, cells carrying a Kint2::ura4+ reporter, in whichthe cenH nucleation region is unaltered, did not show de-fects in silencing (Figure S2A). These results indicate thatPds5 is required for the maintenance of heterochromatin atthe mat locus.

Pds5 and RNAi play distinct roles inheterochromatic silencing

RNAi machinery plays an important role in the nucleation ofheterochromatin and gene silencing (Hall et al. 2002; Volpeet al. 2002; Verdel et al. 2004). To understand the relation-ship between Pds5 and the RNAi machinery, we deleted Pds5in a strain lacking the RNAi factor Ago1 and looked for effectson silencing of the Kint2::ura4+ reporter inserted at cenH.Compared to the single ago1D or pds5D deletionmutants, theago1D pds5D double mutant showed severe loss-of-silencingof Kint2::ura4+ (Figure S2C). Moreover, double mutantsshowed a cumulative increase in transcripts originating fromcenH (Figure S2D). A similar result was obtained when weanalyzed the expression of centromeric repeats in single anddouble mutants. Whereas loss of Pds5 alone causedonly minor changes in the expression of dg centromeric re-peats, the combination of pds5D with ago1D resulted in acumulative increase in dg transcripts that was comparableto that of clr4D cells (Figure S3). These results are consistentwith Pds5 and RNAi performing distinct roles in promotingsilencing at centromeres and the mat locus.

Localization of Pds5 across heterochromatin domainsrequires Swi6

We next asked whether Pds5 acts directly to facilitate hetero-chromatic silencing. Pds5associateswith the cohesin complex(Tanaka et al. 2001; Wang et al. 2002; Schmidt et al. 2009)and localizes to centromeres in a manner dependent on

Figure 2 Pds5 is required for heterochroma-tin formation at the mat locus. (A) Iodinestaining of single colonies. The indicatedstrains were grown in YEA, replica plated ontoPMG5S, and grown at 32� for 3 days prior tostaining. (B) H3K9me2 and H3K9me3 ChIP-qPCR analysis of indicated loci in WT andpds5D cells. The euchromatic locus fbp1serves as a control. The percentage of inputthat was immunoprecipitated is shown (% In-put). The position of the oligonucleotides isindicated in the schematic above. Errorbars denote SEM; N = 3 independent strains.* P , 0.05, ** P , 0.01, and **** P ,0.0001 (Student’s t-test). (C) RT-qPCR analysisof mat2-Pc and ura4+ genes in the indicatedstrains is shown as the relative fold enrichmentover act1. Error bars denote SEM; N $ 3 in-dependent experiments. * P, 0.05 (Student’st-test). ChIP, chromatin immunoprecipitation;PMG5S, pombe minimal glutamate plus fivesupplements; qPCR, quantitative PCR; WT,wild-type; YEA, yeast extract plus adenine.


cohesin (Tanaka et al. 2001). However, cohesin localizationat heterochromatin locations remains unaffected in the ab-sence of Pds5 (Yamagishi et al. 2010). Consistent with theselocus-specific studies, our analyses revealed that Pds5 is lo-calized in the nucleus and is enriched throughout heterochro-matin domains, including at centromeres, telomeres, and thesilent mat region (Figure 5, A–D). Also, we noted a remark-able genome-wide colocalization of Pds5 with the cohesinsubunits Rad21 and Psc3 (Mizuguchi et al. 2014; Folcoet al. 2017) (Figure S4, A–C). In addition to their identicaldistribution profiles within heterochromatin domains, Pds5and cohesin subunits colocalized at the 39-ends of convergentgenes (Figure 5E and Figure S4C).

We previously reported that Swi6 recruits the cohesin-loading complex to heterochromatic regions (Fischer et al.2009). Cells lacking Clr4, which is required for Swi6 locali-zation, therefore fail to load cohesin to heterochromatin do-mains (Bernard et al. 2001; Nonaka et al. 2002; Schmidt et al.2009; Folco et al. 2017). We wondered whether the hetero-chromatin machinery is also required for Pds5 localization.ChIP-chip analyses revealed that loss of Swi6 or Clr4 causedsevere defects in Pds5 localization at centromeres, and at the

silent mat region (Figure 5, A–D). However, the localizationof Pds5 to euchromatic locations was unaffected in hetero-chromatin-deficient cells (Figure 5E), consistent with previ-ous studies showing that heterochromatin factors aredispensable for cohesin localization at euchromatic regions(Schmidt et al. 2009; Mizuguchi et al. 2014; Folco et al.2017). The finding that Pds5 mimics cohesin localizationsuggested a close connection between these proteins, whichcould reveal the cause of silencing defects in pds5D cells.

Pds5-associated Eso1 is required forheterochromatic silencing

Wenext tested factors associated with Pds5 for a possible rolein heterochromatic silencing. Previous studies have shownthat Pds5 interacts with conserved factors Hrk1, Wpl1, andEso1 to achieve proper sister chromatid cohesion and chro-mosome segregation (Goto et al. 2017). Hrk1, a haspin-re-lated kinase, phosphorylates histone H3 Thr3 to recruitthe chromosomal passenger complex (CPC) (Yamagishiet al. 2010). Wpl1 is an anticohesion factor that promotescohesion loss with or without cohesin release from DNA(Kueng et al. 2006; Feytout et al. 2011; Birot et al. 2017).

Figure 3 Pds5 is required for genome-wideheterochromatin formation. (A) Distributionof H3K9me2 along ChrI in WT and pds5Dcells as determined by ChIP-chip. (B) ChIP-chip analysis of H3K9me2 enrichment atcentromere 1 and tel1L is shown. The foldenrichment of H3K9me2 (y-axis) is plotted atthe indicated chromosome position (x-axis).(C) ChIP-qPCR analysis of H3K9me2 andH3K9me3 enrichment at dg repeats, andthe control fbp1 locus, in WT and pds5D.The percentage of input that was immuno-precipitated is shown (% Input). Error barsdenote SEM; N = 3 independent strains pergenotype. ChIP, chromatin immunoprecipi-tation; ChrI, chromosome I; qPCR, quantita-tive PCR; WT, wild-type.


On the other hand, Eso1 is an acetyltransferase that acety-lates two conserved lysine residues (K105 and K106) withinthe globular head domain of the cohesin subunit Psm3 topromote proper cohesion establishment (Tanaka et al.2000; Skibbens 2009).

To uncover clues to the underlying mechanism, we askedwhether the loss of any of the Pds5-associated factors causessilencing defects similar to those in pds5D. Deletion of thenonessential gene hrk1 (Yamagishi et al. 2010) did not affectmat2P::ura4+ repression and failed to trigger noticeable lev-els of haploid meiosis (Figure 6A). Thus, Pds5-mediated re-cruitment of the CPC has no apparent role in heterochromaticsilencing. Next, we focused on Wpl1 and Eso1. Because of itsessential function in promoting sister chromatid cohesion,cells lacking Eso1 are not viable (Tanaka et al. 2000). How-ever, due to their antagonistic roles in cohesion establish-ment, the lethality of eso1D can be suppressed by deletion

of wpl1 (Feytout et al. 2011; Kagami et al. 2011). Whereaswpl1D did not show defects in heterochromatic silencing,the eso1D wpl1D double mutant showed derepression ofmat2P::ura4+ and haploid meiosis similar to pds5D cells(Figure 6A). Moreover, when we deleted pds5 in cellslacking Eso1 and/or Wpl1, the levels of haploid meiosisdisplayed by double or triple mutants were comparable tothat of single-mutant pds5D (Figure S5). Taken together,these results indicate that the cohesin acetyltransferaseEso1 is the Pds5-associated factor that is critical for hetero-chromatic silencing.

Acetylation-mimicking cohesin mutations mitigateheterochromatin defects in pds5D cells

We considered that the lack of acetylation of cohesin maybe the major cause of heterochromatin defects observed inpds5D cells. To directly test this, we asked if a mutation in the

Figure 4 Pds5 is a heterochromatin mainte-nance factor. (A) Examples of haploidgrowth phenotypes obtained from tetraddissection analysis of KD::ade6+ “OFF” 3pds5D crosses. Dissection plates were replicaplated onto PMG – ADE and YE (low ADEplates). (B) ChIP-qPCR analysis of H3K9me3enrichment at indicated loci in WT andpds5D cells. Data are shown as relative foldenrichment compared to the control fbp1locus. The position of the oligonucleotidesis indicated in the schematic in (A). Error barsdenote SEM; N = 3 independent strainsper genotype. * P , 0.05, ** P , 0.01,and *** P , 0.001 (Student’s t-test). ADE,adenine; ChIP, chromatin immunoprecipitation;PMG, pombe minimal glutamate; qPCR, quan-titative PCR; WT, wild-type.


cohesin subunit Psm3 that mimics acetylation by Eso1 canrestore heterochromatin assembly in eso1D or pds5D cells.We combined the psm3K105QK106Q (psm3KK/QQ) mutantallele with eso1D, eso1D wpl1D, or pds5D. As expected,psm3KK/QQ suppressed the lethality caused by eso1D (Figure6B) (Feytout et al. 2011; Kagami et al. 2011). Notably, we

found that eso1D psm3KK/QQ showed neither haploid meio-sis nor derepression of mat2P::ura4+, indicating that thepsm3KK/QQ acetylation-mimicking mutation can preventheterochromatin defects caused by loss of Eso1 (Figure6B). Further underscoring a potentially important require-ment for Psm3 acetylation, the psm3KK/QQ allele rescued

Figure 5 Pds5 localizes throughout heterochromatin do-mains. (A) Microscopy analysis of Pds5-GFP localization inWT, swi6D, and clr4D cells. On the bottom, fluorescenceand brightfield images are merged. (B–E) ChIP-chip analysisof Pds5 distribution. Pds5-GFP localization along ChrII (B),centromere 1 (C), the mat locus (D), and a euchromaticchromosomal arm region (E) is shown for the indicatedstrains. The fold enrichment of Pds5 (y-axis) is plotted atthe indicated chromosome position (x-axis). Green barsrepresent open reading frames according to the 2007 -S. pombe genome assembly. ChIP, chromatin immunopre-cipitation; ChrI/II, chromosome I/II; WT, wild-type.


defective heterochromatic silencing in eso1D wpl1D andpds5D (Figure 6B). In the case of pds5D psm3KK/QQ, cellsgrew on counterselective FOA medium, indicating mainte-nance ofmat2P::ura4+ silencing, but still exhibited some de-gree of haploid meiosis (Figure 6, B and C).

We next explored whether restoration of silencing bypsm3KK/QQ is linked to heterochromatin formation. Remark-ably, pds5D cells carrying the psm3KK/QQ allele showed amarked increase in H3K9me2/3 levels at the silent mat re-gion (Figure 6D). Together, these results suggest that defec-tive heterochromatic silencing in pds5D cells is functionallyconnected to the lack of cohesin acetylation by the Eso1acetyltransferase.

Cohesin removal prevents heterochromatin defects inpds5D cells

As defective cohesin acetylation is linked to the phenotypesobserved in pds5D, we wondered if cohesin itself is requiredfor heterochromatic silencing. We tested this possibility byassaying the effects of psc3-4T and rad21-K1 cohesin mutants

on silencing at the mat locus. Cohesin mutants displayed nei-ther haploidmeiosis nor derepression ofmat2P::ura4+ (Figure7A). This finding is consistent with previous work suggestingthat cohesin is dispensable for heterochromatic silencing(Nonaka et al. 2002; Yamagishi et al. 2008). However, a pos-sibility remained that residual levels of cohesin in the partialloss-of-function psc3-4T and rad21-K1mutants might be suffi-cient to support heterochromatic silencing. To rule this out, wecreated a strain lacking cohesin specifically at heterochromaticlocations (Figure 7B). We took advantage of a previous obser-vation that lethality caused by loss of mitotic cohesin subunitsPsc3 and Rad21 can be bypassed by overexpression of meioticcohesin subunits Rec11 and Rec8 (Kitajima et al. 2003). Im-portantly, Rec8 and Rec11 specifically localize to chromosomalarms but not to heterochromatic regions. Indeed, our ChIPanalyses showed that Rec8 is enriched at chromosomal armregions, but not at the silentmat region and at pericentromericregions (Figure 7C). As expected, cells lacking cohesin at cen-tromeres were sensitive to the spindle poison thiabendazole(Figure 7B).Whenwe used this strain to assay silencing at the

Figure 6 Lack of cohesin acetylation impairsheterochromatic silencing. (A and B) Ten-fold serial dilutions of the indicated strainswere spotted on YEA, with or withoutFOA, and PMG5S minimal media, and grownfor 3 days at 30 and 32�, respectively.PMG5S plates were stained with iodine va-por. (C) Iodine staining of single coloniesgenerated from the indicated strains. Cellswere grown on YEA at 30�, replica platedonto PMG5S minimal media, and grown at32� for 3 days prior to staining with iodinevapor. (D) H3K9me2 and H3K9me3 ChIP-qPCR analyses of the indicated loci areshown as the percentage of immunoprecipi-tated input (% Input) in pds5D and pds5Dpsm3KK/QQ. The position of the oligonucle-otides is shown in the schematic above. Errorbars denote SEM; N = 3 independent strains.* P, 0.05 (Student’s t-test). ChIP, chromatinimmunoprecipitation; PMG5S, pombe mini-mal glutamate plus five supplements; qPCR,quantitative PCR; WT, wild-type; YEA, yeastextract plus adenine.


mat locus, we found that depletion of cohesin had no impact.Indeed, unlike pds5D, cells lacking cohesin at the mat locusshowed no haploid meiosis (Figure 7D).

Our results suggested that cohesin itself is dispensable forheterochromatic silencing. However, the fact that a mutationmimicking cohesin acetylation can restore silencing in pds5Dcells (Figure 6, B–D) indicated that the unacetylated form ofcohesin may impede heterochromatin formation. One predic-tion of this reasoning is that defective heterochromatin as-sembly in pds5D cells could be prevented by removingcohesin at heterochromatic loci. To test this, we deletedpds5 in cells lacking cohesin at the silentmat region. Remark-ably, the absence of cohesin suppressed the heterochromatic

silencing defect in pds5D (Figure 7, D and E). Moreover, thisspecific loss of cohesin from heterochromatic loci in pds5Dcells resulted in a marked increase in H3K9me3 levels at thesilent mat region as compared to the pds5D mutant (Figure7F). These results support the conclusion that in cells lackingPds5 and its associated Eso1, the unacetylated form of cohe-sin interferes with the proper assembly of heterochromaticstructures.

Discussion

Previous studies using the genetically tractable S. pombemodel system have led to valuable insights into conserved

Figure 7 Loss of cohesin suppresses haploidmeiosis in pds5D. (A) Left: ten-fold serial di-lutions of the indicated strains were spottedon YEA rich media, with or without additionof FOA, and grown at 30�. Right: iodinestaining of single colonies generatedfrom the indicated strains. Cells weregrown on YEA at 26�, replica plated ontoPMG5S minimal media, and grown at 32�for 4 days prior to staining with iodine vapor.(B) Top: schematic of mitotic and meioticcohesin complexes (Kitajima et al. 2003).Bottom: ten-fold serial dilutions of the indi-cated strains were spotted on YEA rich me-dia, with or without the spindle poison TBZ,and grown at 32�. (C) ChIP-qPCR analysis ofRec8-HA enrichment at individual loci in theindicated strains. Data are shown as relativefold enrichment compared to the fbp1 con-trol locus. Error bars denote SD; N = 2 inde-pendent experiments. Mean values markedwith different letters (a or b) indicate resultsthat are significantly different from eachother, as established by one-way ANOVAand Holm–Sidak test for multiple compari-sons, respectively (P, 0.05). (D) Iodine stain-ing of single colonies generated from theindicated strains. Cells were grown on YEAat 32�, replica plated onto EMM minimalmedia, and grown at 30� for 3 days priorto staining with iodine vapor. (E) RT-qPCRanalysis of mat2-Pc in the indicated strainsis shown as the relative fold enrichmentcompared to the control leu1 locus. Errorbars denote SD; N = 2 independent experi-ments. One-way ANOVA followed by Holm–Sidak test (P , 0.01). (F) ChIP-qPCR analysisof H3K9me3 enrichment at the indicated lociin pds5D cells expressing either mitotic or mei-otic cohesin. The percentage of input thatwas immunoprecipitated is shown (% Input).Error bars denote SEM; N = 9 independentexperiments. ** P , 0.01 and *** P ,0.001 (Student’s t-test). ChIP, chromatin im-munoprecipitation; FOA, 5-flouroorotic acid;PMG5S, pombe minimal glutamate plus fivesupplements; qPCR, quantitative PCR; TBZ,thiabendazole; WT, wild-type; YEA, yeast ex-tract plus adenine.


heterochromatin assembly pathways (Grewal and Jia 2007;Allshire and Madhani 2018). Indeed, . 50 proteins havebeen shown to contribute to the assembly of heterochromatindomains. In particular, unbiased genetic screens have identi-fied a variety of factors that impact nucleation, spreading,and/or epigenetic inheritance of heterochromatin. However,significant gaps remain in our understanding of the underly-ing mechanisms.

In this study, we used a highly sensitive reporter system toperform a systematic screen for factors that affect heterochro-matic silencing at the silent mat locus. In addition to identi-fying previously known heterochromatin assembly factors,such as components of the CLRC (Hong et al. 2005; Hornet al. 2005; Jia et al. 2005), HP1 proteins (Thon andVerhein-Hansen 2000; Sadaie et al. 2004), SHREC (Thonand Klar 1992; Ekwall and Ruusala 1994; Thon et al. 1994;Sugiyama et al. 2007), FACT (facilitates chromatin transcrip-tion) (Lejeune et al. 2007), and Fft3 (Strålfors et al. 2011;Taneja et al. 2017), we identified eight additional factors.Among the newly identified factors were 60S ribosomal pro-teins (Rpl2102, Rpl39, and Rpl502) and the 19S proteasomeregulatory subunit Rpn10. While it remains unclear how ri-bosomal proteins impact heterochromatic silencing, it is pos-sible that loss of Rpn10 increases levels of antisilencingfactors that affect the spreading and epigenetic inheritanceof heterochromatin. Indeed, levels of Epe1, which negativelyaffects heterochromatin stability (Ayoub et al. 2003), are reg-ulated by the protein degradation machinery (Braun et al.2011). Moreover, a mutation in the Rpt4 subunit of the 19Sproteasome was reported to affect heterochromatin spread-ing (Seo et al. 2017). However, the major finding from thiswork is that the cohesin-associated factor Pds5 is required forthe stable maintenance of heterochromatin. Our analysessuggest that Pds5 prevents the cohesin protein complex,which binds preferentially across heterochromatin domains(Bernard et al. 2001; Nonaka et al. 2002), from interferingwith heterochromatin assembly.

Pds5 localizes throughout heterochromatin domains andits loss causes defective silencing that correlates with reducedH3K9me levels. Evidence suggests thatPds5 is required for thestable propagation of heterochromatin. Indeed, deletion ofpds5 in cells lacking Ago1, which is required for RNAi-mediatednucleation of heterochromatin, causes a cumulative de-crease in H3K9me levels. How might Pds5 contribute toheterochromatic silencing? Considering that Pds5 forms acomplex with cohesin that has been shown to play an impor-tant role in chromosome architecture (Mizuguchi et al. 2014;Kim et al. 2016), it is possible that pds5D cells are defective incohesin-dependent higher-order chromatin organization atheterochromatic loci. However, several lines of evidence sug-gest that, with the exception of a rad21 mutant that displaysreduced heterochromatin formation at subtelomeric regions(Dheur et al. 2011), cohesin itself is dispensable for hetero-chromatic silencing. First, heterochromatin is not affected incells carrying thermosensitive mutations in cohesin subunits(Nonaka et al. 2002) (this study). Second, we find that cells

specifically devoid of cohesin at themat locus are proficient inheterochromatin assembly and gene silencing. Third, it hasbeen shown that artificial recruitment of cohesin to hetero-chromatic loci in cells lacking Swi6 is not sufficient totrigger gene silencing (Yamagishi et al. 2008). Thus, whilePds5–cohesin may contribute to higher-order organization atheterochromatic loci, it is unlikely that defects in such organi-zation are responsible for the silencing phenotype displayed bypds5D cells.

As described above, Pds5 interacts with multiple factors tocoordinate diverse chromosomal events. In addition to recruit-ing Hrk1 kinase and the CPC, which are involved in sisterchromatid biorientation during cell division, Pds5 associateswith Eso1 acetyltransferase and Wpl1, which are linked tocohesin dynamics (Vaur et al. 2012; Goto et al. 2017). Amongthese, we find that Eso1 is themost critical factor for the role ofPds5 in heterochromatin maintenance, although silencing atthe edge of a heterochromatin domain at the mat locus hasbeen shown to be affected in cells lacking Wpl1 (Jahn et al.2018). Given our results showing that an acetylation-mimick-ing cohesinmutant can suppress silencing defects displayed byeso1D and pds5D, it is likely that the lack of cohesin acetylationcreates a major impediment to heterochromatin assembly.Acetylated cohesin might limit the effects of antisilencing fac-tors such as Epe1 (Ayoub et al. 2003), which is also recruitedby Swi6 (Zofall and Grewal 2006). Another possibility is thatunacetylated cohesin may directly interfere with heterochro-matin assembly. This second possibility is supported by ourfinding that eliminating cohesin from heterochromatic loca-tions can restore the proper maintenance of heterochromatinin cells lacking Pds5.

Cohesin acetylation is a central determinant of replicationfork processivity in mammalian systems (Terret et al. 2009;Sherwood et al. 2010), and loss of Pds5 can hinder replica-tion progression (Carvajal-Maldonado et al. 2019). More-over, depletion of Rad21 in a Pds5-deficient backgroundrescues the replication defect (Carvajal-Maldonado et al.2019). Considering that factors required for DNA replicationalso impact heterochromatin maintenance (Nakayama et al.2001a; Jahn et al. 2018), it is possible that defects in hetero-chromatin formation caused by loss of Pds5 and its associatedEso1 might be linked to impaired replication. Changes inreplication might affect the preservation of parental modifiedhistones required for Clr4 loading, which in turn epigeneti-cally maintains heterochromatin domains through its abilityto both read and write H3K9me nucleosomes (Zhang et al.2008; Aygün et al. 2013). Regardless of the mechanism, ourfindings highlight an important role for the conserved Pds5protein in coordinating interplay between cohesin involvedin sister chromatid cohesion and factors contributing to het-erochromatin maintenance.

Acknowledgments

We thank Takeshi Sakuno (Osaka University, Japan) and theNational BioResource Project-Yeast (Japan) for strains, Jemima


Barrowman for editing the manuscript, Martin Zofall andSahana Holla for technical assistance, and members of theGrewal laboratory for discussions. This work was supportedby the Intramural Research Program of the NationalInstitutes of Health, National Cancer Institute.

Author contributions: H.D.F. and S.I.S.G. conceived theproject, and designed experiments. H.D.F. performed mostof the experiments including the genome-wide screen. A.M.constructed strains, and performed RT-PCR and ChIP-chipexperiments. V.B. constructed strains. All authors contrib-uted to data interpretation. H.D.F. and S.I.S.G. wrote themanuscript with input from all authors.

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cohesin impedes heterochromatin assembly in fission yeast ... · an acetylation-mimicking mutation...

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