Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin
Ontology highlight
ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE10561 | GEO | 2008/03/16
SECONDARY ACCESSION(S): PRJNA107815
REPOSITORIES: GEO
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Project description:ChIP-chip analyses of ClrC components across the S. pombe genome. Keywords: ChIP-chip Chromatin immunoprecipitated DNA recovered with antibody against FLAG-tagged protein from wild-type asynchronous growing fission yeast cells and whole cell extracts DNA were amplified by random priming and respectively labeled with Cy5 and Cy3. Labeled DNA samples were mixed and hybridized onto a 44k pombe Agilent oligo array. Data were processed using Agilent scanner and Feature Extraction software
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Project description:Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6(HP1) are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6(HP1) operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.
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Project description:Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.
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Project description:Expression profile of mutant cells compared to wild-type cells: Expression profile of Schizosaccharomyces pombe genome in ∆pht1, ∆ago1, ∆clr4, rik1, ∆pht1rik1, ∆pht1∆ago1 and ∆pht1∆clr4 cells. Expression profile of Schizosaccharomyces pombe genome in ∆rrp6, ∆cid14, ∆swi6, ∆swr1, ∆set1, ∆pht1∆swi6 and ∆pht1∆set1 cells. Expression profile of Schizosaccharomyces pombe genome in ∆alp13, ∆cph1, ∆set2, clr6-1 and ∆cph1∆pht1 cells. Occupancy profiling: Occupancy profiling of RNA polymerase II, histone variant H2A.Z and ClrC subunit Rik1 in fission yeast Schizosaccharomyces pombe Occupancy profiling of histone variant H2A.Z in ∆msc1 cells.
2009-08-25 | GSE17271 | GEO
Project description:Expression profile of mutant cells compared to wild-type cells: Expression profile of Schizosaccharomyces pombe genome in ∆pht1, ∆ago1, ∆clr4, rik1, ∆pht1rik1, ∆pht1∆ago1 and ∆pht1∆clr4 cells. Expression profile of Schizosaccharomyces pombe genome in ∆rrp6, ∆cid14, ∆swi6, ∆swr1, ∆set1, ∆pht1∆swi6 and ∆pht1∆set1 cells. Expression profile of Schizosaccharomyces pombe genome in ∆alp13, ∆cph1, ∆set2, clr6-1 and ∆cph1∆pht1 cells. Occupancy profiling: Occupancy profiling of RNA polymerase II, histone variant H2A.Z and ClrC subunit Rik1 in fission yeast Schizosaccharomyces pombe Occupancy profiling of histone variant H2A.Z in ∆msc1 cells. Agilent 60mer oligonucleotide custom array containing probes spanning large portion of chromosome 2 at 50bp resolution was used to profile expression levels in mutant cells and to compare them to levels in wild-type cells. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of RNA polymerase II, H2A.Z or Rik1 from asynchronous culture of fission yeast. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of histone H2A.Z from asynchronous culture of fission yeast.
2009-08-25 | E-GEOD-17271 | biostudies-arrayexpress
Project description:Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.
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Project description:To investigate the role of Clr4 recruitment, we performed FLAG-Clr4 ChIP-seq in various genetic background
2024-01-01 | GSE223202 | GEO
Project description:Heterochromatin formation in fission yeast depends on RNAi machinery and histone-modifying enzymes. One of the key histone-modifying complexes is Clr4-Rik1-Cul4 methyltransferase complex (CLRC), which mediates histone H3K9 methylation, a hallmark for heterochromatin. CLRC is composed of the Clr4 histone methyltransferase, Rik1, Raf1, Raf2 and Pcu4. However, transcriptional regulation of the CLRC subunits is not well understood. In this study, we identified Set3, a core subunit of the Set3/Hos2 histone deacetylase complex (Set3C), as a contributor to the integrity and silencing of heterochromatin at centromeres, telomeres and silent mating-type locus. This novel role of Set3 relies on its PHD finger, but is independent of deacetylase activity or structural integrity of Set3C. Set3 is not located to the centromeric region. Instead, Set3 is targeted to the promoters of clr4(+) and rik1(+), probably through its PHD finger. Set3 promotes transcription of clr4(+) and rik1(+). Consistently, the protein levels of Clr4 and Rik1 were reduced in the set3? mutant. The heterochromatin silencing defect in the set3? mutant could be rescued by overexpressing of clr4(+) or rik1(+). Our study suggests transcriptional activation of essential heterochromatin factors underlies the tight regulation of heterochromatin integrity.
| S-EPMC4990937 | biostudies-literature
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.
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Project description:We report global occupancy of H3K9me2 and H3K4me3 in wild-type S. pombe and two mutant backgrounds - the deletion of heterochromatin antagonist epe1 and the double mutant absent epe1 and the H3K4 methyltransferase set1
2020-08-24 | GSE140067 | GEO