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• W2171319591 abstract "Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 functions in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promotes nucleosome occupancy to assemble repressive heterochromatin. Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 functions in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promotes nucleosome occupancy to assemble repressive heterochromatin. S. pombe Asf1 binds histones and HIRA that localizes across heterochromatin domains Asf1/HIRA and Clr6 HDAC act in concert to enforce heterochromatic silencing Asf1 and SHREC promote nucleosome occupancy at heterochromatic loci Asf1 facilitates global histone deacetylation and suppresses spurious transcription Eukaryotic genomes are in general organized into two types of chromatin: euchromatin and heterochromatin. Whereas euchromatin is transcriptionally competent, heterochromatin is typically transcriptionally repressed. The factors involved in heterochromatin assembly are found broadly distributed across genomes, but their main targets are chromosomal regions containing high density of repetitive DNA such as transposons and their remnants found at centromeres and telomeres (Grewal and Jia, 2007Grewal S.I.S. Jia S. Heterochromatin revisited.Nat. Rev. Genet. 2007; 8: 35-46Crossref PubMed Scopus (932) Google Scholar). Heterochromatin promotes genomic stability by exerting repressive influence on the expression of parasitic transposable elements and by prohibiting the illegitimate recombination between dispersed repetitive DNA elements (Peng and Karpen, 2008Peng J.C. Karpen G.H. Epigenetic regulation of heterochromatic DNA stability.Curr. Opin. Genet. Dev. 2008; 18: 204-211Crossref PubMed Scopus (140) Google Scholar). Heterochromatin assembly involves posttranslational modifications of histones and a common set of structural proteins. With the exception of budding yeast, heterochromatin assembly requires methylation of histone H3 at lysine 9 (H3K9me) that provides binding sites for HP1 family of chromodomain proteins (Jenuwein and Allis, 2001Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7206) Google Scholar). In the fission yeast Schizosaccharomyces pombe, Clr4 not only methylates H3K9 to create binding sites for the localization of chromodomain proteins Chp1, Chp2, and Swi6 (Bannister et al., 2001Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.Nature. 2001; 410: 120-124Crossref PubMed Scopus (2059) Google Scholar, Nakayama et al., 2001Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I.S. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science. 2001; 292: 110-113Crossref PubMed Scopus (1308) Google Scholar, Sadaie et al., 2004Sadaie M. Iida T. Urano T. Nakayama J. A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast.EMBO J. 2004; 23: 3825-3835Crossref PubMed Scopus (166) Google Scholar, Schalch et al., 2009Schalch T. Job G. Noffsinger V.J. Shanker S. Kuscu C. Joshua-Tor L. Partridge J.F. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.Mol. Cell. 2009; 34: 36-46Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) but also binds to H3K9me via its own chromodomain to promote the spreading of heterochromatin (Zhang et al., 2008Zhang K. Mosch K. Fischle W. Grewal S.I.S. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin.Nat. Struct. Mol. Biol. 2008; 15: 381-388Crossref PubMed Scopus (243) Google Scholar). Heterochromatin factors are preferentially enriched across the chromosomal domains containing specific repeats, referred to as dg and dh repeats, that are present at pericentromeric regions, subtelomeres and the silent mating-type (mat) locus (Cam et al., 2005Cam H.P. Sugiyama T. Chen E.S. Chen X. FitzGerald P.C. Grewal S.I.S. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.Nat. Genet. 2005; 37: 809-819Crossref PubMed Scopus (375) Google Scholar). RNA polymerase II (RNAPII) transcribes the dg/dh repeats, but their expression is repressed by heterochromatin. Chp1, a subunit of the RITS (RNA-induced transcriptional silencing) complex containing Ago1 and Tas3 proteins, docks RNAi machinery to heterochromatin, where RITS and its associated factors degrade repeat transcripts, thus causing posttranscriptional silencing in cis (cis-PTGS) (Noma et al., 2004Noma K. Sugiyama T. Cam H. Verdel A. Zofall M. Jia S. Moazed D. Grewal S.I.S. RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing.Nat. Genet. 2004; 36: 1174-1180Crossref PubMed Scopus (312) Google Scholar, Schalch et al., 2009Schalch T. Job G. Noffsinger V.J. Shanker S. Kuscu C. Joshua-Tor L. Partridge J.F. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.Mol. Cell. 2009; 34: 36-46Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (911) Google Scholar). Similarly, Chp2 and Swi6 provide recruiting platform for factors involved in transcriptional gene silencing (TGS). The localization of SHREC, which contains a class II HDAC Clr3 and an Snf2 family protein Mit1, across heterochromatin domains requires Chp2 and Swi6 (Sugiyama et al., 2007Sugiyama T. Cam H.P. Sugiyama R. Noma K. Zofall M. Kobayashi R. Grewal S.I.S. SHREC, an effector complex for heterochromatic transcriptional silencing.Cell. 2007; 128: 491-504Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Yamada et al., 2005Yamada T. Fischle W. Sugiyama T. Allis C.D. Grewal S.I.S. The nucleation and maintenance of heterochromatin by a histone deacetylase in fission yeast.Mol. Cell. 2005; 20: 173-185Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Swi6 also associates with class I HDAC Clr6 that acts broadly to mediate the global deacetylation of histones, including at RNAPII-transcribed regions (Nicolas et al., 2007Nicolas E. Yamada T. Cam H.P. Fitzgerald P.C. Kobayashi R. Grewal S.I.S. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.Nat. Struct. Mol. Biol. 2007; 14: 372-380Crossref PubMed Scopus (137) Google Scholar). Clr3 and Clr6 as well as their interacting HP1 proteins act in an overlapping manner to mediate heterochromatic TGS (Fischer et al., 2009Fischer T. Cui B. Dhakshnamoorthy J. Zhou M. Rubin C. Zofall M. Veenstra T.D. Grewal S.I.S. Diverse roles of HP1 proteins in heterochromatin assembly and functions in fission yeast.Proc. Natl. Acad. Sci. USA. 2009; 106: 8998-9003Crossref PubMed Scopus (98) Google Scholar). Mutations in Clr3 and Mit1 subunits of SHREC affect nucleosome positioning that correlates with the TGS defects (Sugiyama et al., 2007Sugiyama T. Cam H.P. Sugiyama R. Noma K. Zofall M. Kobayashi R. Grewal S.I.S. SHREC, an effector complex for heterochromatic transcriptional silencing.Cell. 2007; 128: 491-504Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Heterochromatin assembly also requires histone chaperones (Eitoku et al., 2008Eitoku M. Sato L. Senda T. Horikoshi M. Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly.Cell. Mol. Life Sci. 2008; 65: 414-444Crossref PubMed Scopus (146) Google Scholar). Among the chaperones that deliver histones to DNA, CAF-1 (chromatin assembly factor 1) and HIRA (histone regulatory homolog A) mediate DNA replication-dependent and -independent chromatin assembly, respectively (Groth et al., 2007bGroth A. Rocha W. Verreault A. Almouzni G. Chromatin challenges during DNA replication and repair.Cell. 2007; 128: 721-733Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, Ransom et al., 2010Ransom M. Dennehey B.K. Tyler J.K. Chaperoning histones during DNA replication and repair.Cell. 2010; 140: 183-195Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). CAF-1 interacts with HP1 and is required for the replication and the maintenance of heterochromatin (Murzina et al., 1999Murzina N. Verreault A. Laue E. Stillman B. Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins.Mol. Cell. 1999; 4: 529-540Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, Quivy et al., 2004Quivy J.P. Roche D. Kirschner D. Tagami H. Nakatani Y. Almouzni G. A CAF-1 dependent pool of HP1 during heterochromatin duplication.EMBO J. 2004; 23: 3516-3526Crossref PubMed Scopus (146) Google Scholar). HIRA is involved in silencing heterochromatic loci (Greenall et al., 2006Greenall A. Williams E.S. Martin K.A. Palmer J.M. Gray J. Liu C. Whitehall S.K. Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation.J. Biol. Chem. 2006; 281: 8732-8739Crossref PubMed Scopus (45) Google Scholar, Kaufman et al., 1998Kaufman P.D. Cohen J.L. Osley M.A. Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I.Mol. Cell. Biol. 1998; 18: 4793-4806Crossref PubMed Scopus (145) Google Scholar, Sharp et al., 2001Sharp J.A. Fouts E.T. Krawitz D.C. Kaufman P.D. Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing.Curr. Biol. 2001; 11: 463-473Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, Ye et al., 2007Ye X. Zerlanko B. Zhang R. Somaiah N. Lipinski M. Salomoni P. Adams P.D. Definition of pRB- and p53-dependent and -independent steps in HIRA/ASF1a-mediated formation of senescence-associated heterochromatin foci.Mol. Cell. Biol. 2007; 27: 2452-2465Crossref PubMed Scopus (124) Google Scholar). Both CAF-1 and HIRA cooperate with a ubiquitous histone chaperone Asf1 (antisilencing factor 1), which is believed to deliver histones H3 and H4 heterodimer for nucleosome assembly (Ransom et al., 2010Ransom M. Dennehey B.K. Tyler J.K. Chaperoning histones during DNA replication and repair.Cell. 2010; 140: 183-195Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Loss of Asf1 causes sensitivity to genotoxic agents (Tyler et al., 1999Tyler J.K. Adams C.R. Chen S.R. Kobayashi R. Kamakaka R.T. Kadonaga J.T. The RCAF complex mediates chromatin assembly during DNA replication and repair.Nature. 1999; 402: 555-560Crossref PubMed Scopus (432) Google Scholar). However, the exact cause of this phenotype is not fully understood. In this study, we define Asf1 functions in heterochromatic silencing and global protective functions of chromatin in S. pombe. Asf1 forms complex with histones H3 and H4 as well as HIRA that spreads across heterochromatin domains in a manner dependent upon Swi6/HP1. Asf1 associates with a Clr6 complex, and these factors act in concert to mediate large-scale deacetylation of histones, including at euchromatic loci. This function of Asf1 is essential to suppress the intragenic antisense transcripts and to protect the DNA from damage. We demonstrate that Asf1 and SHREC function in overlapping pathways impacting nucleosome occupancy at heterochromatic loci. Thus, Asf1 contributes to chromatin structural modulations by facilitating histone deacetylation and governing nucleosome occupancy, which has important implications for the assembly and the propagation of repressive heterochromatin. We previously showed that amino-terminal TAP-tagged Swi6 copurifies with factors involved in chromosome dynamics and heterochromatic silencing (Fischer et al., 2009Fischer T. Cui B. Dhakshnamoorthy J. Zhou M. Rubin C. Zofall M. Veenstra T.D. Grewal S.I.S. Diverse roles of HP1 proteins in heterochromatin assembly and functions in fission yeast.Proc. Natl. Acad. Sci. USA. 2009; 106: 8998-9003Crossref PubMed Scopus (98) Google Scholar). Mass spectrometry of purified Swi6 samples also identified peptides corresponding to HIRA subunit Hip3 (see Figure S1A available online). These results indicated that HIRA might directly participate in heterochromatic silencing. Considering that Asf1 synergizes with HIRA and CAF-1 to assemble nucleosomes (Tagami et al., 2004Tagami H. Ray-Gallet D. Almouzni G. Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis.Cell. 2004; 116: 51-61Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, Tyler et al., 1999Tyler J.K. Adams C.R. Chen S.R. Kobayashi R. Kamakaka R.T. Kadonaga J.T. The RCAF complex mediates chromatin assembly during DNA replication and repair.Nature. 1999; 402: 555-560Crossref PubMed Scopus (432) Google Scholar), we wondered whether these factors act together in heterochromatin assembly. To test this, we purified carboxy-terminal TAP-tagged Asf1 (Asf1-TAP) using tandem affinity purification (TAP). Mass spectrometry of the purified samples showed that Asf1 associate with histones H3 and H4 as well as HIRA comprising Hip1, Slm9, Hip3, and Hpc2 (Figure 1A and Figure S1B). This analysis did not identify CAF-1 in the Asf1 purified fraction (Figure 1A). To address this further, we purified carboxy-terminal TAP-tagged Pcf3 (p48) subunit of CAF-1. In addition to Pcf3, we identified Pcf1 (p150) and Pcf2 (p60) subunits of CAF-1, as well as few peptides of Pcn1 (PCNA) (Figure 1A and Figure S1C), which associates with CAF-1 (Shibahara and Stillman, 1999Shibahara K. Stillman B. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin.Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), but no Asf1 peptides. It is possible that Asf1 interacts with CAF-1 in a cell cycle stage-specific manner (e.g., during S phase) that is not detected in our assays. Regardless, Asf1 interactions with histones H3/H4 and HIRA are conserved in S. pombe, thus, raising the possibility of these factors acting together, in association with HP1, to enforce heterochromatic silencing. We next investigated whether Asf1 affects heterochromatic silencing. Since Asf1 is essential for growth in S. pombe (Umehara et al., 2002Umehara T. Chimura T. Ichikawa N. Horikoshi M. Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro.Genes Cells. 2002; 7: 59-73Crossref PubMed Scopus (56) Google Scholar), we constructed a mutant allele of asf1 (asf1-1). Mutant Asf1 shows a considerable reduction in its ability to bind histones and HIRA (Figure 1B). Sequencing of asf1-1 showed one mutation mapping to a residue involved in histone binding, while another mapped close to HIRA-binding site (Figure S2A). We tested the effects of asf1-1 on silencing at heterochromatic loci (Figure S2B). For comparison, we included deletion mutants of genes encoding HIRA subunits. asf1-1 alleviated silencing of the ura4+ inserted at the outer centromeric repeat region (otr1R::ura4+) and within a centromere-homologous (cenH) element at the silent mat locus (Kint2::ura4+), in a manner similar to HIRA null mutants (Figure 1C) (Greenall et al., 2006Greenall A. Williams E.S. Martin K.A. Palmer J.M. Gray J. Liu C. Whitehall S.K. Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation.J. Biol. Chem. 2006; 281: 8732-8739Crossref PubMed Scopus (45) Google Scholar). Since the growth-based silencing assay is not quantitative, we evaluated silencing defects by quantitative real-time RT-PCR (qPCR). Effects of asf1-1 and HIRA null mutants on expression of ura4+ reporters and heterochromatic repeats at centromeres (dg/dh) and mat locus (cenH) were examined (Figure 1D). Also included in this analysis were Tf2 retrotransposons. Tf2 silencing requires HIRA, SHREC, and Clr6, but not Clr4 and HP1 (Cam et al., 2008Cam H.P. Noma K. Ebina H. Levin H.L. Grewal S.I.S. Host genome surveillance for retrotransposons by transposon-derived proteins.Nature. 2008; 451: 431-436Crossref PubMed Scopus (125) Google Scholar, Greenall et al., 2006Greenall A. Williams E.S. Martin K.A. Palmer J.M. Gray J. Liu C. Whitehall S.K. Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation.J. Biol. Chem. 2006; 281: 8732-8739Crossref PubMed Scopus (45) Google Scholar, Hansen et al., 2005Hansen K.R. Burns G. Mata J. Volpe T.A. Martienssen R.A. Bahler J. Thon G. Global effects on gene expression in fission yeast by silencing and RNA interference machineries.Mol. Cell. Biol. 2005; 25: 590-601Crossref PubMed Scopus (107) Google Scholar). The asf1-1 and HIRA mutants showed severe defects in the silencing of Tf2. However, these mutations only partially derepressed heterochromatic repeats or ura4+ reporters, as compared to the clr4Δ (Figure 1D). Clr4 is required for both TGS and cis-PTGS of heterochromatic loci (Noma et al., 2004Noma K. Sugiyama T. Cam H. Verdel A. Zofall M. Jia S. Moazed D. Grewal S.I.S. RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing.Nat. Genet. 2004; 36: 1174-1180Crossref PubMed Scopus (312) Google Scholar). Transcripts that escape heterochromatic TGS are processed into siRNAs by RNAi machinery. asf1-1 and HIRA mutants only affect TGS and not cis-PTGS. Indeed, we observed increased levels of siRNAs in asf1-1 and HIRA mutants (Figure 1E), revealing that dg/dh repeats are derepressed in these mutants but transcripts are not accumulating due to their degradation by RNAi. Heterochromatin also regulates mating-type switching by promoting nonrandom choice of mat2 and mat3 donor loci (Jia et al., 2004Jia S. Yamada T. Grewal S.I.S. Heterochromatin regulates cell type-specific long-range chromatin interactions essential for directed recombination.Cell. 2004; 119: 469-480Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). S. pombe spores, but not the vegetative cells, stain dark with iodine, while the switching-defective mutants produce spores inefficiently, resulting in light staining. Defective mating-type switching in heterochromatin mutants results in poor iodine staining of colonies, in contrast to the dark staining of wild-type colonies (Jia et al., 2004Jia S. Yamada T. Grewal S.I.S. Heterochromatin regulates cell type-specific long-range chromatin interactions essential for directed recombination.Cell. 2004; 119: 469-480Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). asf1-1 and HIRA mutants were defective in mating-type switching, as indicated by the light iodine staining of the colonies (Figure 1F). Therefore, Asf1 contributes not only to silencing but also other functions that rely on heterochromatin. HP1 proteins serve as a recruiting platform to target silencing activities such as SHREC (Sugiyama et al., 2007Sugiyama T. Cam H.P. Sugiyama R. Noma K. Zofall M. Kobayashi R. Grewal S.I.S. SHREC, an effector complex for heterochromatic transcriptional silencing.Cell. 2007; 128: 491-504Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Yamada et al., 2005Yamada T. Fischle W. Sugiyama T. Allis C.D. Grewal S.I.S. The nucleation and maintenance of heterochromatin by a histone deacetylase in fission yeast.Mol. Cell. 2005; 20: 173-185Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Asf1/HIRA might affect silencing by facilitating the assembly of H3K9me-HP1 platform, or it could act downstream of HP1 loading as an effector involved in repressive chromatin assembly. We used chromatin immunoprecipitation (ChIP) assay to examine the effects of asf1-1 and hip1Δ on levels of H3K9me and HP1 proteins at centromeres. Whereas asf1-1 cells showed slight reduction in levels of H3K9me, Swi6, and Chp2, the levels of these factors in hip1Δ appeared comparable to wild-type (Figures 2A and 2B ). Thus, silencing defects in asf1-1 and hip1Δ were not coupled to complete loss of the heterochromatin platform. The reduction in H3K9me is not due to defective Clr4 recruitment but rather reflects dynamic nature of nucleosomes in mutant cells (see below). We next investigated whether HP1 proteins contribute to the localization of histone chaperone at heterochromatic loci. Since we detected Hip3 in Swi6 purified fraction (Figure S1A), we wondered if HIRA associates with heterochromatin machinery. Hip1 was readily detected in the affinity-purified Swi6 fraction (Figure 2C). This interaction is functionally important because Swi6 was required for Hip1 distribution across the heterochromatic domains. ChIP-chip showed that Hip1 was enriched throughout heterochromatin domains in the wild-type cells (Figures 2D and 2E, and Figure S3). In the absence of Swi6, Hip1 localization was restricted to transcribed dg/dh repeats, and it failed to spread outward to the surrounding sequences (Figures 2D and 2E, and Figure S3). Defects in spreading of Hip1 in swi6Δ mutant could be seen clearly at silent mat locus and telomeres, which unlike centromeres contain a single copy of repeat element and require Swi6 for spreading heterochromatin across the silenced domain (Cam et al., 2005Cam H.P. Sugiyama T. Chen E.S. Chen X. FitzGerald P.C. Grewal S.I.S. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.Nat. Genet. 2005; 37: 809-819Crossref PubMed Scopus (375) Google Scholar, Hall et al., 2002Hall I.M. Shankaranarayana G.D. Noma K. Ayoub N. Cohen A. Grewal S.I.S. Establishment and maintenance of a heterochromatin domain.Science. 2002; 297: 2232-2237Crossref PubMed Scopus (683) Google Scholar, Kanoh et al., 2005Kanoh J. Sadaie M. Urano T. Ishikawa F. Telomere binding protein Taz1 establishes Swi6 heterochromatin independently of RNAi at telomeres.Curr. Biol. 2005; 15: 1808-1819Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). These results suggest that Hip1 can be targeted to dg/dh elements independent of Swi6, but its localization across the heterochromatin domains requires Swi6. Asf1/HIRA influence heterochromatic silencing directly by associating with Swi6/HP1. To address the underlying mechanism, we explored the effects of asf1 and hip1 mutants in combination with the mutant alleles of other silencing activities such as RNAi RITS (tas3), SHREC (clr3 and mit1), and Clr6 complex (clr6 and alp13). The effects of single and double mutants on the levels of transcripts derived from dg/dh and cenH elements were assayed using qPCR. Consistent with both TGS and cis-PTGS contributing to heterochromatin silencing, combining asf1-1 or hip1Δ with tas3Δ resulted in synergistic defects in heterochromatic silencing (Figure 3A ). We also found that double mutants carrying mutations in clr3 or mit1 along with either asf1-1 or hip1Δ showed cumulative derepression of repeat elements (Figure 3A and Figure S4), indicating overlapping functions for Asf1/HIRA and SHREC. However, when asf1-1 or hip1Δ were combined with clr6 or alp13 mutant alleles, double mutants did not show additive defects on silencing as compared to the single mutants (Figure 3A and Figure S4). This result argues that Asf1/HIRA and Clr6 HDAC act in concert to silence heterochromatic repeats. Based on the genetic analyses, it was possible that Asf1/HIRA facilitate histone deacetylation by Clr6. Asf1 coimmunoprecipitated with Clr6 complex subunits Alp13 and Clr6 (Figure 3B). Moreover, asf1-1 and hip1Δ exhibited a substantial increase in bulk H3K9ac levels, in a manner similar to alp13Δ (Figure 3C). To confirm this further, we performed ChIP-chip analyses of H3K9ac. Both alp13Δ and asf1-1 mutants showed widespread increase in H3K9ac, as compared to the wild-type cells. Notably, although 30% of the probes in our microarray correspond to intergenic regions, nearly all probes affected by asf1-1 and alp13Δ reside in coding regions (Figures 3D and 3E). These data are in agreement with the previous conclusion that Alp13-containing Clr6 complex (referred to as Clr6 complex II) deacetylates histones preferentially in transcribed regions of the genome (Nicolas et al., 2007Nicolas E. Yamada T. Cam H.P. Fitzgerald P.C. Kobayashi R. Grewal S.I.S. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.Nat. Struct. Mol. Biol. 2007; 14: 372-380Crossref PubMed Scopus (137) Google Scholar). Closer examination of the data revealed that all genomic sites that showed increased histone acetylation in asf1 mutant were also affected by Clr6 complex II, except the partial loss of function asf1-1 allele in general showed weaker effects as compared to alp13Δ. Changes in histone acetylation prompted us to analyze transcriptional changes in asf1 mutant. For comparison, we also included alp13Δ and hip1Δ that show upregulation of antisense RNA (Anderson et al., 2009Anderson H.E. Wardle J. Korkut S.V. Murton H.E. Lopez-Maury L. Bahler J. Whitehall S.K. The fission yeast HIRA histone chaperone is required for promoter silencing and the suppression of cryptic antisense transcripts.Mol. Cell. Biol. 2009; 29: 5158-5167Crossref PubMed Scopus (48) Google Scholar, Nicolas et al., 2007Nicolas E. Yamada T. Cam H.P. Fitzgerald P.C. Kobayashi R. Grewal S.I.S. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.Nat. Struct. Mol. Biol. 2007; 14: 372-380Crossref PubMed Scopus (137) Google Scholar). Expression profiling detected widespread upregulation of transcripts in all three mutant backgrounds. The affected loci included Tf2 retrotransposon and LTRs. Moreover, asf1-1 and hip1Δ showed upregulation of sense and antisense transcripts corresponding to subtelomeric genes located within heterochromatic domains (Figure S5A). Interestingly, asf1-1, but not hip1Δ, also showed substantial increase in the levels of transcripts derived from intergenic portions of rDNA repeat loci (Figure S5B). Notably, asf1-1 produced a disproportionate increase in antisense transcripts—constituting a large proportion of probes upregulated. Detailed expression profiling of individual loci showed that the antisense transcripts upregulated in asf1-1 mutants were also upregulated in hip1Δ and alp13Δ cells (Figures 4A and 4B ). The appearance of antisense transcripts in mutant backgrounds was confirmed by strand-specific RT-PCR (Figure 4C). Clr4 complex and RNAi factors facilitate the degradation of readthrough antisense RNA at euchromatic loci via a mechanism involving nuclear exosome (Zofall et al., 2009Zofall M. Fischer T. Zhang K. Zhou M. Cui B. Veenstra T.D. Grewal S.I.S. Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs.Nature. 2009; 461: 419-422Crossref PubMed Scopus (115) Google Scholar). This mechanism is distinct from a pathway involving Clr6 and Set2 that suppresses initiation of antisense transcript from cryptic promoters (Nicolas et al., 2007Nicolas E. Yamada T. Cam H.P. Fitzgerald P.C. Kobayashi R. Grewal S.I.S. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.Nat. Struct. Mol. Biol. 2007; 14: 372-380Crossref PubMed Scopus (137) Google Scholar). To gain further insight into Asf1 functions, we compared the distributions of antisense RNA in different mutants. Correlation coefficients from pairwise comparisons were used to cluster mutants based on similarity in their antisense profiles (Figure 4D). Antisense profiles of asf1-1 and hip1Δ mutants closely resembled to that of cells lacking Clr6 complex components Alp13 and Cph1, or Set2 involved in H3K36me at tr" @default.
• W2171319591 created "2016-06-24" @default.
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• W2171319591 date "2011-01-01" @default.
• W2171319591 modified "2023-09-30" @default.
• W2171319591 title "Asf1/HIRA Facilitate Global Histone Deacetylation and Associate with HP1 to Promote Nucleosome Occupancy at Heterochromatic Loci" @default.
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• W2171319591 doi "https://doi.org/10.1016/j.molcel.2010.12.009" @default.
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