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Abo1 is required for H3K9me2 to H3K9me3 transition in telomeric and centromeric heterochromatin [ChIP-seq]

ABSTRACT: Regulation of heterochromatin is critical for genome stability. Different states of methylated H3K9 have been discovered with distinct roles in heterochromatin formation and silencing. However, the control of the transition from H3K9me2 to H3K9me3 is still unclear. Here we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining the global nucleosome organization in fission yeast. We identified several key factors involved in heterochromatin silencing to interact genetically with Abo1: the histone deacetylase Clr3, the H3K9 methyltransferase Clr4, and the HP1 homologue Swi6. Cells lacking Abo1 display an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin had increased H3K9me2 but decreased H3K9me3 levels compared to wild type. In contrast, facultative heterochromatin regions, show both reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both centromeres and subtelomeres, but not in a subset of heterochromatin islands. Our work uncovers a new role for Abo1 in supporting Clr4 activity and allowing for the transition of H3K9me2 to H3K9me3 in telomeric and centromeric heterochromatin.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE125911 | GEO | 2020/04/13

REPOSITORIES: GEO

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Project description:Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in chromatin-dependent gene silencing and maintenance of genome stability by repressing repetitive DNA elements and recruiting downstream factors essential for faithful chromosome segregation. Fundamental principles of these processes have been elucidated in the fission yeast Schizosaccharomyces pombe (S. pombe), in which Clr4, the homologue of mammalian SUV39H, is the sole H3K9 methyltransferase. Although H3K9 methylation has been intensely studied in mitotic cells, occurrence and regulation during sexual differentiation remain largely unknown. Whether distinct H3 methylation states are relevant for gametogenesis is also not known. Here, using diploid S. pombe cells, we map H3K9 methylation genome-wide during the meiotic program and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells exit mitosis and commit to the meiotic life cycle. Cells lacking the ability to tri-methylate H3K9 exhibit severe meiotic chromosome segregation defects, which does not occur in mitotic cells. Artificially increasing the affinity of the fission yeast heterochromatin protein 1 (HP1) homologue Swi6 towards H3K9me2 rescues chromosome segregation in the absence of H3K9me3. Thus, the H3K9me2 to H3K9me3 switch during early meiosis serves to retain Swi6 at centromeres, which is necessary for cohesin recruitment and kinetochore assembly. Finally, we show that the H3K9 methylation switch is regulated by differential phosphorylation of Clr4 by the cyclin-dependent kinase (CDK) Cdk1. Our results reveal how a highly conserved master regulator of the cell cycle regulates the specificity of the H3K9 methyltransferase and thereby controls the ratio of H3K9me2 to H3K9me3 to prevent unwanted gene silencing and to ensure faithful meiotic chromosome segregation. High degree of conservation of the proteins involved in our study and CDK-dependent phosphorylation of the mammalian SUV39H1 homologue suggest broad conservation of the mechanisms described here.

Project description:Heterochromatic DNA domains play important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. In organisms ranging 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 transcribed from DNA repeats, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation. H3K9me is in turn required for recruitment of RNAi to chromatin and promotes sRNA generation. However, 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 homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations, which allow H3K9me2 catalysis but block the transition to H3K9me3. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS). 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, 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 heterochromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in RNAi-mediated genome defense.

Project description:Heterochromatin plays a key role in gene repression, maintaining genome integrity and chromosome segregation. Fission yeast, Schizosaccharomyces pombe, utilizes conserved components to direct heterochromatin formation using siRNA generated by RNA interference (RNAi) to guide a histone H3 lysine 9 methyltransferase to cognate chromatin. To identify compounds that inhibit heterochromatin formation, an in vivo phenotypic screen for loss of silencing was performed. A tester strain harbouring a silent dominant selectable kanMX reporter gene within fission yeast centromeric heterochromatin was used to screen a diverse library of chemicals. HMS-I1 and HMS-I2 were identified as compounds that reproducibly increased G418 resistance due to loss of kanMX silencing, and decreased the level of repressive H3K9 methylation on centromeric repeats. The pattern of changes induced by HMS-I1 and HMS-I2 were consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic interactions and expression profiling indicated that both HMS-I1 and HMS-I2 affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). Exposure to HMS-I1 was also found to alleviate silencing of reporter genes in an Arabidopsis transgenic plant line and a mouse cell line. HMS-I2 also disrupted reporter gene silencing in Arabidopsis. In vitro assays indicate that HMS-I1 impairs the activity of human HDAC6 and HDAC10. As HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities they represent potentially novel and valuable reagents for experimental and therapeutic purposes. Our findings highlight the use of in vivo chemical screening conducted in fission yeast to identify compounds that disrupt heterochromatin across plant, fungi and animal kingdoms. 16 RNA samples: 2 replicates of WT untreated (vehicle DMSO) (KE1-A, KE2-A), HMS-I1 treated (KE1-B, KE2-B), HMS-I2 treated (KE1-C, KE2-C), and 2 additional replicates of WT (KE05_wt_1, KE10_wt_2), 4 replicates of clr2M-bM-^HM-^F (KE01_2118_1, KE06_2118_2, KE03_0080_1, KE08_0080_2) and mit1M-bM-^HM-^F (KE02_1295_1, KE07_1295_2, KE04_1278_1, KE09_1278_2).

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Abo1 is required for H3K9me2 to H3K9me3 transition in telomeric and centromeric heterochromatin [ChIP-seq]

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