Mechanism for the selective sequestration of a histone H3K9 methyltransferase at heterochromatin by the H3K9M mutation
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PROVIDER: PRJNA667923 | ENA |
REPOSITORIES: ENA
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Project description:Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild type histones, but the mechanisms by which these mutations function are highly controversial. We have previously shown that in fission yeast, H3K9M containing nucleosomes sequesters the H3K9 methyltransferase Clr4 to block H3K9 methylation{Shan, 2016 #119}. Using ChIP-sequencing, here we show that even though H3K9M containing nucleosomes are broadly distributed across the genome, Clr4 is mainly sequestered at pericentric repeats. The 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 enhances the interaction between H3K9M and Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Using bio-layer interferometry, we show that the H3K9M mutation reduced association rates (kon) of Clr4 with histone tails, suggesting that Clr4 intrinsically disfavors H3K9M. More importantly, H3K9M reduced the dissociation rate (koff) of Clr4 with histone tails, demonstrating that H3K9M slows down the turnover of Clr4 once it is bound. Finally, we generated mutations on Clr4 that specifically disrupt its interaction with H3K14ub and show that they reduce the inhibitory effects of H3K9MK14ub on Clr4 enzymatic activity in vitro and relieve the sequestration of Clr4 at pericentric repeats in vivo. Therefore, the selective sequestration of Clr4 at pericentric repeats by H3K9M is due to the presence of H3K14ub at these regions, which overcomes Clr4’s aversion of H3K9M and reduces the dissociation of Clr4.
2020-10-26 | GSE159192 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all 6 SET domain methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEFs no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is the first analysis of H3K9 methylation deficient mammalian chromatin and reveals a crucial function for H3K9 methylation in protecting heterochromatin organization and genome integrity.
2021-05-27 | PXD018175 | Pride
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224639 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224638 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224640 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224636 | GEO
Project description:Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions, and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of compound mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me2/3, was redistributed to regions targeted by H3K9me2/3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that the two major repressive epigenome pathways exclusively but also coordinately maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.
2022-04-05 | GSE200014 | GEO
Project description:Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions, and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of compound mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me2/3, was redistributed to regions targeted by H3K9me2/3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that the two major repressive epigenome pathways exclusively but also coordinately maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.
2022-04-05 | GSE200015 | GEO
Project description:Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions, and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of compound mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me2/3, was redistributed to regions targeted by H3K9me2/3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that the two major repressive epigenome pathways exclusively but also coordinately maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.
2022-04-05 | GSE200012 | GEO
Project description:Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions, and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of compound mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me2/3, was redistributed to regions targeted by H3K9me2/3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that the two major repressive epigenome pathways exclusively but also coordinately maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.
2022-04-05 | GSE200011 | GEO