Histone chaperone Spt16p is required for heterochromatin mediated silencing in budding yeast.
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ABSTRACT:
SUBMITTER: Yan X
PROVIDER: S-EPMC6019661 | biostudies-literature | 2018 Jul
REPOSITORIES: biostudies-literature
Publications
Histone chaperone Spt16p is required for heterochromatin mediated silencing in budding yeast.
Yan Xiaowei X Yang Jiayi J Xu Jiawei J Feng Jianxun J Li Qing Q
Protein & cell 20180701 7
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Project description:Epigenetic inheritance of heterochromatin structure is an important cellular process whose mechanism remains elusive. In this article, we describe the identification of nine enhancers of the silencing defect of a Saccharomyces cerevisiae-PCNA mutant by screening a library of approximately 4,700 viable yeast deletion mutants. Of the nine mutants identified, six (hir1, hir3, sas2, sas4, sas5, and sir1) were previously known to reduce silencing synergistically with a mutation in Cac1p, the large subunit of chromatin assembly factor 1 (CAF-1). The predicted gene products that are affected in three other mutants (nam7, msh2, and rtt106) have not been implicated previously in silencing. Characterization of the rtt106Delta allele revealed that it synergistically reduced heterochromatin silencing when combined with a mutation in Cac1p but not with a mutation in Asf1p (a histone H3 and H4 chaperone). Moreover, Rtt106p interacted with histones H3 and H4 both in vitro and in vivo, and it displayed a nucleosome assembly activity in vitro. Furthermore, Rtt106p interacts with CAF-1 physically through Cac1p. These biochemical and genetic data indicate that Rtt106p is a previously uncharacterized histone chaperone connecting S phase to epigenetic inheritance.
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Project description:Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here, we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.
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Project description:Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts (S. cerevisiae and S. pombe), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10. In fission yeast, the RNAi-dependent pathway initiates heterochromatin in the context of low H2B-Ub. We examine how the different silencing machineries overcome the challenge of H2B-Ub chromatin and highlight the importance of using these microorganisms to further our understanding of H2B-Ub in heterochromatic silencing pathways.
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Project description:The assembly of nucleosomes by histone chaperones is an important component of transcriptional regulation. Here, we have assessed the global roles of the HIRA histone chaperone in Schizosaccharomyces pombe. Microarray analysis indicates that inactivation of the HIRA complex results in increased expression of at least 4% of fission yeast genes. HIRA-regulated genes overlap with those which are normally repressed in vegetatively growing cells, such as targets of the Clr6 histone deacetylase and silenced genes located in subtelomeric regions. HIRA is also required for silencing of all 13 intact copies of the Tf2 long terminal repeat (LTR) retrotransposon. However, the role of HIRA is not restricted to bona fide promoters, because HIRA also suppresses noncoding transcripts from solo LTR elements and spurious antisense transcripts from cryptic promoters associated with transcribed regions. Furthermore, the HIRA complex is essential in the absence of the quality control provided by nuclear exosome-mediated degradation of illegitimate transcripts. This suggests that HIRA restricts genomic accessibility, and consistent with this, the chromosomes of cells lacking HIRA are more susceptible to genotoxic agents that cause double-strand breaks. Thus, the HIRA histone chaperone is required to maintain the protective functions of chromatin.
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Project description:In eukaryotes, heterochromatin regions are typically subjected to transcriptional silencing. DNA methylation has an important role in such silencing and has been studied extensively. However, little is known about how methylated heterochromatin regions are subjected to silencing. We conducted a genetic screen and identified an epcr (enhancer of polycomb-related) mutant that releases heterochromatin silencing in Arabidopsis thaliana We demonstrated that EPCR1 functions redundantly with its paralog EPCR2 and interacts with PWWP domain-containing proteins (PWWPs), AT-rich interaction domain-containing proteins (ARIDs), and telomere repeat binding proteins (TRBs), thus forming multiple functionally redundant protein complexes named PEAT (PWWPs-EPCRs-ARIDs-TRBs). The PEAT complexes mediate histone deacetylation and heterochromatin condensation and thereby facilitate heterochromatin silencing. In heterochromatin regions, the production of small interfering RNAs (siRNAs) and DNA methylation is repressed by the PEAT complexes. The study reveals how histone deacetylation, heterochromatin condensation, siRNA production, and DNA methylation interplay with each other and thereby maintain heterochromatin silencing.
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Project description:Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regulation. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.
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Project description:In eukaryotes, heterochromatin regions are typically subjected to transcriptional silencing. DNA methylation has an important role in such silencing, and has been studied extensively. However, little is known about how methylated heterochromatin regions are subjected to silencing. We conducted a genetic screen and identified an epcr (enhancer of polycomb-related) mutant that releases heterchromatin silencing in Arabidopsis thaliana. We demonstrated that EPCR1 functions redundantly with its paralog EPCR2 and interacts with PWWP domain-containing proteins (PWWPs), AT-rich interaction domain-containing proteins (ARIDs), and telomere repeat binding proteins (TRBs), thus forming multiple functionally redundant protein complexes named PEAT (PWWPs-EPCRs-ARIDs-TRBs). The PEAT complexes mediate histone deacetylation and heterochromatin condensation and thereby facilitate heterochromatin silencing. In heterochromatin regions, the production of small interfering RNAs (siRNAs) and DNA methylation are repressed by the PEAT complexes. The study reveals how histone deacetylation, heterochromatin condensation, siRNA production, and DNA methylation interplay with each other and thereby maintain heterochromatin silencing.
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Project description:Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin factors results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication and repair by anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications that modify nucleosome dynamics, such as H2B ubiquitylation (H2Bub). We report here that H2Bub and Asf1 cooperatively promote transcriptional silencing at yeast telomeres and mating loci. Through real time monitoring of HML (Hidden MAT Left) locus silencing, we found that transcriptional repression was slowly initiated and never fully established in mutants lacking both Asf1 and H2Bub. These findings are consistent with impaired HML silencer-binding and spreading of repressor proteins, Sir2 and Sir3. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing at mating loci. Thus, the interplay between H2Hbub and Asf1 may fine-tune nucleosome dynamics and SIR protein recruitment, and represent an ongoing requirement for proper formation and maintenance of heterochromatin.
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Project description:Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.
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Project description:Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.
2019-11-28 | PXD015484 | Pride