RNA interference guides histone modification during the S phase of chromosomal replication.
ABSTRACT: Heterochromatin is chromosomal material that remains condensed throughout the cell division cycle and silences genes nearby. It is found in almost all eukaryotes, and although discovered (in plants) almost 100 years ago, the mechanism by which heterochromatin is inherited has remained obscure. Heterochromatic silencing and histone H3 lysine-9 methylation (H3K9me2) depend, paradoxically, on heterochromatic transcription and RNA interference (RNAi).Here, we show that heterochromatin protein 1 in fission yeast (Swi6) is lost via phosphorylation of H3 serine 10 (H3S10) during mitosis, allowing heterochromatic transcripts to transiently accumulate in S phase. Rapid processing of these transcripts into small interfering RNA (siRNA) promotes restoration of H3K9me2 and Swi6 after replication when cohesin is recruited. We also show that RNAi in fission yeast is inhibited at high temperatures, providing a plausible mechanism for epigenetic phenomena that depend on replication and temperature, such as vernalization in plants and position effect variegation in animals.These results explain how "silent" heterochromatin can be transcribed and lead to a model for epigenetic inheritance during replication.
Project description:HP1 proteins are a highly conserved family of eukaryotic proteins that bind to methylated histone H3 lysine 9 (H3K9) and are required for heterochromatic gene silencing. In fission yeast, two HP1 homologs, Swi6 and Chp2, function in heterochromatic gene silencing, but their relative contribution to silencing remains unknown. Here we show that Swi6 and Chp2 exist in nonoverlapping complexes and make distinct contributions to silencing. Chp2 associates with the SHREC histone deacetylase complex (SHREC2), is required for histone H3 lysine 14 (H3K14) deacetylation, and mediates transcriptional repression by limiting RNA polymerase II access to heterochromatin. In contrast, Swi6 associates with a different set of nuclear proteins and with noncoding centromeric transcripts and is required for efficient RNAi-dependent processing of these transcripts. Our findings reveal an unexpected role for Swi6 in RNAi-mediated gene silencing and suggest that different HP1 proteins ensure full heterochromatic gene silencing through largely nonoverlapping inhibitory mechanisms.
Project description:Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.
Project description:Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin. H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.
Project description:Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A(Cnp1) and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A(Cnp1) chromatin on naïve templates. Once assembled, CENP-A(Cnp1) chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified.
Project description:Heterochromatin silencing is critical for genomic integrity and cell survival. It is orchestrated by chromodomain (CD)-containing proteins that bind to methylated histone H3 lysine 9 (H3K9me), a hallmark of heterochromatin. Here, we show that phosphorylation of tyrosine 41 (H3Y41p)-a novel histone H3 modification-participates in the regulation of heterochromatin in fission yeast. We show that a loss-of-function mutant of H3Y41 can suppress heterochromatin de-silencing in the centromere and subtelomere repeat regions, suggesting a de-silencing role for H3Y41p on heterochromatin. Furthermore, we show both in vitro and in vivo that H3Y41p differentially regulates two CD-containing proteins without the change in the level of H3K9 methylation: it promotes the binding of Chp1 to histone H3 and the exclusion of Swi6. H3Y41p is preferentially enriched on centromeric heterochromatin during M- to early S phase, which coincides with the localization switch of Swi6/Chp1. The loss-of-function H3Y41 mutant could suppress the hypersensitivity of the RNAi mutants towards hydroxyurea (HU), which arrests replication in S phase. Overall, we describe H3Y41p as a novel histone modification that differentially regulates heterochromatin silencing in fission yeast via the binding of CD-containing proteins.
Project description:Histone modification marks have an important role in many chromatin processes. During DNA replication, both heterochromatin and euchromatin are disrupted ahead of the replication fork and are then reassembled into their original epigenetic states behind the fork. How histone marks are accurately inherited from generation to generation is still poorly understood. In fission yeast (Schizosaccharomyces pombe), RNA interference (RNAi)-mediated histone methylation is cell cycle regulated. Centromeric repeats are transiently transcribed in the S phase of the cell cycle and are processed into short interfering RNAs (siRNAs) by the complexes RITS (RNA-induced initiation of transcriptional gene silencing) and RDRC (RNA-directed RNA polymerase complex). The small RNAs together with silencing factors-including Dos1 (also known as Clr8 and Raf1), Dos2 (also known as Clr7 and Raf2), Rik1 and Lid2-promote heterochromatic methylation of histone H3 at lysine 9 (H3K9) by a histone methyltransferase, Clr4 (refs 8-13). The methylation of H3K9 provides a binding site for Swi6, a structural and functional homologue of metazoan heterochromatin protein 1 (HP1). Here we characterize a silencing complex in fission yeast that contains Dos2, Rik1, Mms19 and Cdc20 (the catalytic subunit of DNA polymerase-?). This complex regulates RNA polymerase II (RNA Pol II) activity in heterochromatin and is required for DNA replication and heterochromatin assembly. Our findings provide a molecular link between DNA replication and histone methylation, shedding light on how epigenetic marks are transmitted during each cell cycle.
Project description:Heterochromatin is a conserved feature of eukaryotic genomes and regulates various cellular processes, including gene silencing, chromosome segregation, and maintenance of genome stability. In the fission yeast Schizosaccharomyces pombe, heterochromatin formation involves methylation of lysine 9 in histone H3 (H3K9), which recruits Swi6/HP1 proteins to heterochromatic loci. The Swi6/HP1-H3K9me3 chromatin complex lies at the center of heterochromatic macromolecular assemblies and mediates many functions of heterochromatin by recruiting a diverse set of regulators. However, additional factors may be required for proper heterochromatin organization, but they are not fully known. Here, using several molecular and biochemical approaches, we report that Vgl1, a member of a large family of multiple KH-domain proteins, collectively known as vigilins, is indispensable for the heterochromatin-mediated gene silencing in S. pombe ChIP analysis revealed that Vgl1 binds to pericentromeric heterochromatin in an RNA-dependent manner and that Vgl1 deletion leads to loss of H3K9 methylation and Swi6 recruitment to centromeric and telomeric heterochromatic loci. Furthermore, we show that Vgl1 interacts with the H3K9 methyltransferase, Clr4, and that loss of Vgl1 impairs Clr4 recruitment to heterochromatic regions of the genome. These findings uncover a novel role for Vgl1 as a key regulator in heterochromatin-mediated gene silencing in S. pombe.
Project description:Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.
Project description: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.
Project description:Proteins of the conserved HP1 family are elementary components of heterochromatin and are generally assumed to play a central role in the creation of a rigid, densely packed heterochromatic network that is inaccessible to the transcription machinery. Here, we demonstrate that the fission yeast HP1 protein Swi6 exists as a single highly dynamic population that rapidly exchanges in cis and in trans between different heterochromatic regions. Binding to methylated H3K9 or to heterochromatic RNA decelerates Swi6 mobility. We further show that Swi6 is largely dispensable to the maintenance of heterochromatin domains. In the absence of Swi6, H3K9 methylation levels are maintained by a mechanism that depends on polymeric self-association properties of Tas3, a subunit of the RNA-induced transcriptional silencing complex. Our results disclose a surprising role for Swi6 dimerization in demarcating constitutive heterochromatin from neighboring euchromatin. Thus, rather than promoting maintenance and spreading of heterochromatin, Swi6 appears to limit these processes and appropriately confine heterochromatin.