KAP1 facilitates reinstatement of heterochromatin after DNA replication
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE86391 | GEO | 2018/07/03
REPOSITORIES: GEO
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Project description:DNA replication ensures the accurate transmission of genetic information during cell cycle. The interaction between histone methyltransferase SUV420H1 and histone variant H2A.Z plays a critical role in the licensing of early replication origins. However, the mechanism by which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on DNA replication origins remains elusive. Here, we determined the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome. Our structures show that the SUV420H1 catalytic domain (CD) directly interacts with histone H4 and nucleosomal DNA, whereas a SUV420H1 arginine-rich motif (ARM) anchors to the acidic patch of the nucleosome. The N-terminal aminal acid residues of H4 (Aa 1-24) forms a lasso-shaped structure sandwiched between SUV420H1 CD and nucleosome. The lasso-shaped structure stabilizes the SUV420H1-nucleosome interaction and precisely projects the H4 K20 residue into the SUV420H1 catalytic center. Further analyses revealed a crucial role of SUV420H1 KR-loop (aminal acid residues 214-223), which spatially lies closely to H2A.Z specific residues D97/S98, in dictating the preference for H2A.Z-nucleosome. SUV420H1 K219A/R220A mutations reduced the activity of SUV420H1 for H4K20me2 modification, the preference of SUV420H1 for H2A.Z-nucleosome, and the efficiency of DNA replication initiation. Collectively, our findings elucidate how SUV420H1 preferentially recognizes H2A.Z-nucleosome to deposit H4K20me2 modification and shed light on therapeutic strategies targeting the DNA replication initiation.
2023-12-04 | GSE210197 | GEO
Project description:KAP1 facilitates reinstatement of heterochromatin after DNA replication
| PRJNA341671 | ENA
Project description:Microarrays analysis identifies WNT16in senescent fibroblasts. To identify genes expressed in replicative senescence, microarray experiments were conducted in which we compared the RNA expression profile of young MRC5 fibroblasts at population doubling 28 (PDL28) and replicative senescent MRC5 fibroblasts at PDL63. In total, 177 gene probes were upregulated and 338 gene probes were downregulated in senescent versus young MRC5 fibroblasts. Genes were classified based on mapping to KEGG pathways. Upregulated genes were enriched in three KEGG pathways, the Wingless-type MMTV integration site (WNT) signaling pathway (WNT16, PLCB4, CCND2, SFRP1, WNT5B, RAC2), cytokine-cytokine receptor interaction (TNFRSF10D, TNFRSF10B, TNFRSF10C, IL1R2, IL18, VEGFC), and the p53 signaling pathway (CDKN1A, TNFRSF10B, PERP, STEAP3, ZMAT3). Repressed genes were enriched in three KEGG pathways, cell cycle (PKMYT1, CDC7, CCNE2, MCM4, BUB1, CDC25A, CDKN1B, MCM6, PLK1, CDKN2C, CCNB2, MCM3, BUB1B, CCNA2, CDC2), cytokine-cytokine receptor interaction (KITLG, IL11, TNFSF4, CCL2, PDGFRA, TNFRSF11B, TGFBR1, IL1R1, TNFRSF19), and DNA replication (POLE, RFC2, MCM4, RNASEH2B, POLA1, DNA2, MCM6, PRIM1, RFC4, MCM3). Along with p53 transcriptional targets (CDKN1A, PERP, WIG1), genes encoding secreted factors (WNT16, MMP10, MMP3) were among the most strongly increased in senescent cells.
2009-12-15 | GSE15919 | GEO
Project description:Microarrays analysis identifies WNT16in senescent fibroblasts. To identify genes expressed in replicative senescence, microarray experiments were conducted in which we compared the RNA expression profile of young MRC5 fibroblasts at population doubling 28 (PDL28) and replicative senescent MRC5 fibroblasts at PDL63. In total, 177 gene probes were upregulated and 338 gene probes were downregulated in senescent versus young MRC5 fibroblasts. Genes were classified based on mapping to KEGG pathways. Upregulated genes were enriched in three KEGG pathways, the Wingless-type MMTV integration site (WNT) signaling pathway (WNT16, PLCB4, CCND2, SFRP1, WNT5B, RAC2), cytokine-cytokine receptor interaction (TNFRSF10D, TNFRSF10B, TNFRSF10C, IL1R2, IL18, VEGFC), and the p53 signaling pathway (CDKN1A, TNFRSF10B, PERP, STEAP3, ZMAT3). Repressed genes were enriched in three KEGG pathways, cell cycle (PKMYT1, CDC7, CCNE2, MCM4, BUB1, CDC25A, CDKN1B, MCM6, PLK1, CDKN2C, CCNB2, MCM3, BUB1B, CCNA2, CDC2), cytokine-cytokine receptor interaction (KITLG, IL11, TNFSF4, CCL2, PDGFRA, TNFRSF11B, TGFBR1, IL1R1, TNFRSF19), and DNA replication (POLE, RFC2, MCM4, RNASEH2B, POLA1, DNA2, MCM6, PRIM1, RFC4, MCM3). Along with p53 transcriptional targets (CDKN1A, PERP, WIG1), genes encoding secreted factors (WNT16, MMP10, MMP3) were among the most strongly increased in senescent cells. Six arrays were hybridized with complex probes consisting of RNA isolated from MRC5 fibroblasts at population doubling 63 (undergoing replicative senescence) and RNA isolated from MRC5 fibroblats at population doubling 28 (young). The six arrays correspond to three biological replicates, each with a dye-swap technical replicate.
2010-05-18 | E-GEOD-15919 | biostudies-arrayexpress
Project description:DNA replication is a tightly regulated process that ensures the precise duplication of the genome during cell cycle. Licensing and activation of eukaryotic replication origins are controlled primarily by chromatin. However, the chromatin features involved and the regulatory mechanism remain largely unknown. In this study, we found that H2A.Z binds Suv420H1 directly to promote H4K20me2 deposition on nucleosome both in vitro and in vivo. ORC1 is subsequently recruited to chromatin for licensing and activation of early replication origins. Depletion of H2A.Z results in defects of DNA replication and cell proliferation in both HeLa cells and T cells. Thus, our results provide novel mechanistic insights that the histone variant H2A.Z epigenetically regulates licensing and activation of the early DNA replication origins through the Suv420H1-H4K20me2-ORC1 pathway.
2019-09-22 | GSE134988 | GEO
Project description:DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2’-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Early and late origins tend to distribute separately in large chromosome regions. Inactivation of replication checkpoint by deletion of Cds1 resulted in BrdU incorporation with HU specifically at the late origins. An origin-exchange experiment showed that replication timing was not intrinsic to origin but dependent on its surrounding region. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. Keywords: ChIP-chip analysis
2007-03-05 | GSE6523 | GEO
Project description:Heterochromatin domains defined by distinct histone methylation patterns are a major feature of mammalian genomes. Epigenetic pathways ensure stable heterochromatin inheritance through cell division to prevent the expression of lineage-inappropriate genes and defects in this process can lead to many diseases including cancer. Previous studies have shown that heterochromatin maintenance requires a critical threshold of methylated histones, but how modified parental histones are transferred to daughter strands during DNA replication is unclear. We find that heterochromatin domains are specialized replication zones enriched in replisome components, which load factors that help retain parental histones. This mechanism is further supported by the boundary elements surrounding the heterochromatin domains. DNA replication is thus coordinated with histone preservation to facilitate epigenetic inheritance of heterochromatin.
2024-02-02 | GSE242353 | GEO
Project description:DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2â-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Early and late origins tend to distribute separately in large chromosome regions. Inactivation of replication checkpoint by deletion of Cds1 resulted in BrdU incorporation with HU specifically at the late origins. An origin-exchange experiment showed that replication timing was not intrinsic to origin but dependent on its surrounding region. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. Keywords: ChIP-chip analysis Experiment Design: ⢠The goal of the experiment â Genome-wide localization of ORC and MCM binding sites and identification of replication origins in Shizosaccaromyces pombe ⢠A brief description of the experiment (e.g., the abstract from the related publication) DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2â-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Early and late origins tend to distribute separately in large chromosome regions. Inactivation of replication checkpoint by deletion of Cds1 resulted in BrdU incorporation with HU specifically at the late origins. An origin-exchange experiment showed that replication timing was not intrinsic to origin but dependent on its surrounding region. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. ⢠Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended). Mitosis, Cell cycle, Schizosaccharomyces pombe, Whole-genome, Tiling array, Chromosome II, III array, Orc1, Orc4, Mcm6, BrdU-labeling, cds1Î. ⢠Experimental factors Distribution of the Orc1 and Mcm6 in WT in G1 phase (S. pombe). Distribution of the Orc4 in WT in asynchronous cells (S. pombe). Distribution of BrdU-incorporated DNA in WT and in cds1 deletion mutant in S phase in the presence of HU (S. pombe). ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Pombe whole-genome tiling array were used. BrdU analysis: Hybridization data for BrdU fraction (replicated DNA) was compared with Whole fraction (unreplicated DNA) or hybridization data for BrdU fraction of cds1Î cells was compared with that of WT cells. Pombe whole-genome tiling array were used. ⢠Quality control steps taken Different subunits of the same complex. Confirmation of several loci by q-PCR. Checking the BrdU-labeled DNA fraction in CsCl gradient by q-PCR or slot-blot analysis. ⢠Links to the publication, any supplemental websites or database accession numbers. GSE6523 Samples used, extract preparation and labelling: ⢠The origin of each biological sample ChIP analysis: Schizosaccharomyces pombe nda3-KM311 cdc10-129 strain harboring pREP81-cdc18 and pREP42-cdt1. BrdU analysis: Schizosaccharomyces pombe wild type (cdc25-22 nmt1-TK) and cds1Î strains. ⢠Manipulation of biological samples and protocols used ChIP analysis: Chromatin immunoprecipitation (ChIP) in G1 arrested cells or asynchronous cells were performed as previously described (Takahashi et al., 2003, EMBO). Hybridization to Affimetrix high-density oligonucleotide array of S. pombe chromosomes 2 and 3 and whole genome tiling array of S. pombe was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). BrdU analysis: Growing cdc25-22 cells expressing Thymidine Kinase were arrested at G2/M boundary and released in the presence of BrdU and HU. BrdU was incorporated in nascent DNA in following S phase. The genomic DNA was digested by HaeIII and BrdU-labeled DNA was purified in CsCl density gradient centrifugation. Hybridization to Affimetrix high-density oligonucleotide array of S. pombe whole genome tiling array was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature).
2010-06-11 | E-GEOD-6523 | biostudies-arrayexpress
Project description:Heterochromatin binding protein HP1β plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1β-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1β is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1β-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1ΔRing cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.
2021-09-24 | PXD025053 | Pride
Project description:Impaired DNA replication is a hallmark of cancer and a cause of genomic instability. We report that, in addition to causing genetic change, impaired DNA replication during embryonic development can have major epigenetic consequences for a genome. In a genome-wide screen, we identified impaired DNA replication as causing increased expression from a repressed transgene in Caenorhabditis elegans. The acquired expression state behaved as an “epiallele,” being inherited for multiple generations before fully resetting. Derepression was not restricted to the transgene but was caused by a global reduction in heterochromatin-associated histone modifications due to the impaired retention of modified histones on DNA during replication in the early embryo. Impaired DNA replication during development can therefore globally derepress chromatin, creating new intergenerationally inherited epigenetic expression states.
2017-09-15 | GSE95034 | GEO