Genome-wide human ORC/ MCM distribution
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB32855 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR3378072.fastq.gz | Fastqsanger.gz | |||
| ERR3392700.fastq.gz | Fastqsanger.gz | |||
| ERR3392710.fastq.gz | Fastqsanger.gz | |||
| ERR3392720.fastq.gz | Fastqsanger.gz | |||
| ERR3392701.fastq.gz | Fastqsanger.gz |
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Project description:Sumoylation is emerging as a post-translation modification important for chromosome duplication and stability. The origin recognition complex (ORC), which directs DNA replication initiation by loading the MCM replicative helicases onto origins, is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are largely unclear. Here we report the effects of hyper- and hypo-sumoylation of yeast ORC using multiple approaches. We show that ORC hyper-sumoylation preferentially reduces the activity of a subset of early origins, while Orc2 hypo-sumoylation has an opposing effect. Mechanistically, ORC hyper-sumoylation leads to reduced MCM loading in vitro and diminished MCM chromatin association in vivo. The importance of an appropriate level of ORC sumoylation is suggested by the data that either hyper- or hypo-sumoylation of ORC results in genome instability and a dependence on other genome maintenance factors for cell fitness. Thus, yeast ORC sumoylation status needs to be fine-tuned to achieve optimal origin activity control and genome stability.
2023-03-11 | PXD032977 | Pride
Project description:Methylation of histone H4 lysine 20 (H4K20), such as H4K20 mono-methylation (H4K20me1), regulates the biological processes of DNA replication, cell cycle progression, and DNA damage repair. Whereas H4K20me1 is knowingly written by SET8 (also known as PR-Set7) and erased by PHF8 (also known as KDM7B), the mechanism underlying H4K20me1-mediated regulation of DNA replication remains murky. Here, we report that a conserved tandem Tudor domain of BAHCC1 (BAHCC1TTD) specifically reads H4K20me1. Our biochemical, structural and cellular analyses demonstrated that the recognition of H4K20me1 by BAHCC1TTD facilitates the BAHCC1 recruitment onto the replication origins, where BAHCC1 interacts and recruits components of Minichromosome Maintenance (MCM) complex, a critical partner machinery of Origin Replication Complex (ORC) for mediating DNA replication. Combined actions of the H4K20me1-reading BAHCC1 and the H4K20me2-reading ORC ensure optimal genomic loading of MCM proteins. Depletion of BAHCC1, or disruption of BAHCC1TTD:H4K20me1 interaction, affected H4K20me1 homeostasis and MCM complex loading, leading to impaired DNA replication and defective cell cycle progression. Together, this study identifies a conserved BAHCC1TTD as an effector linking H4K20me1 to MCM recruitment for efficient DNA replication.
2025-06-03 | GSE213114 | GEO
Project description:We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones (ERIZs) in both mouse and human cells. The identified ERIZs fall in open chromatin compartments while are mutually exclusive with transcription elongation and occupy mainly non-transcribed regions adjacent to transcribed regions. Furthermore, we reveal that RNA polymerase II actively redistributes the chromatin-encircled mini-chromosome maintenance (MCM) complex but not the origin-recognition complex (ORC) to actively restrict early DNA replication initiation outside of transcribed regions. The coupling of RNA polymerase II and MCM is further validated by detected MCM accumulation and DNA replication initiation after RNA polymerase II stalling via anchoring nuclease-dead Cas9 at the transcribed genes. Importantly, we also find that the orchestration of DNA replication initiation by transcription can efficiently prevent gross DNA damage.
2021-05-20 | GSE174680 | GEO
Project description:The hexameric DNA helicase MCM (Mcm2-7) is a central regulatory target in eukaryotic replication. Chromatin-bound MCM is kept inactive during G1 phase and subsequently activated in S phase to initiate replication. During this transition, the only known chemical change on the Mcm2-7 proteins is the gain of multi-site phosphorylation that promotes recruitment of co-factors. As replication initiation is tied intimately to multiple biological cues, additional changes on these proteins can provide a second regulatory point. Here we describe a new MCM modification cycle mediated by SUMO that enables a negative regulation of replication initiation. We show that all MCM subunits undergo sumoylation upon loading at origins in G1 phase prior to MCM phosphorylation. Then bulk MCM sumoylation is lost as MCM phosphorylation rises. The pattern of MCM sumoylation suggests a negative role in replication. Indeed, increasing MCM sumoylation delays genome-wide replication initiation. Mechanistically, this is partly due to enhancing the recruitment of a conserved phosphatase that delays MCM phosphorylation and activation. By revealing a new MCM modification cycle and its role in replication, our findings suggest a new model, in which MCM sumoylation counterbalances kinase-based regulation to ensure accurate control of replication initiation.
2016-02-01 | GSE70407 | 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:The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with several subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decrease ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
2022-10-19 | GSE199896 | GEO
Project description:The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decreased ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
2022-10-19 | PXD033045 | Pride
Project description:Methylation of histone H4 lysine 20 (H4K20), such as H4K20 mono-methylation (H4K20me1), regulates the biological processes of DNA replication, cell cycle progression, and DNA damage repair1-4. Whereas H4K20me1 is knowingly written by SET85,6 (also known as PR-Set7) and erased by PHF87 (also known as KDM7B), the mechanism underlying H4K20me1-mediated regulation of DNA replication remains murky. Here, we report that a conserved tandem Tudor domain of BAHCC1 (BAHCC1TTD) specifically reads H4K20me1. Our biochemical, structural and cellular analyses demonstrated that the recognition of H4K20me1 by BAHCC1TTD facilitates the BAHCC1 recruitment onto the replication origins, where BAHCC1 interacts and recruits components of Minichromosome Maintenance (MCM) complex, a critical partner machinery of Origin Replication Complex (ORC) for mediating DNA replication8,9. Combined actions of the H4K20me1-reading BAHCC1 and the H4K20me2-reading ORC ensure optimal genomic loading of MCM proteins. Depletion of BAHCC1, or disruption of BAHCC1TTD:H4K20me1 interaction, affected H4K20me1 homeostasis and MCM complex loading, leading to impaired DNA replication and defective cell cycle progression. Together, this study identifies a conserved BAHCC1TTD as an effector linking H4K20me1 to MCM recruitment for efficient DNA replication.
2025-06-06 | PXD036436 | Pride
Project description:The minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during G1/S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide deeper understanding towards this event. Here, we uncover that the histidine methyltransferase SETD3 promotes DNA replication in an enzymatic activity dependent manner. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Mechanistically, biochemical experiments reveal that SETD3 binds MCM mainly during G1/S phase, which is required for CDT1-mediated chromatin loading of MCM. The MCM loading relies on the histidine-459 methylation (H459me) on MCM7 that is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.
2024-03-12 | GSE243937 | GEO
Project description:Mono-methylation of histone H4 lysine 20 (H4K20me1) is increasingly appreciated to be critical for regulating genomic DNA replication, cell cycle progression and DNA damage repair. How exactly H4K20me1 regulates these biological processes remains murky. Here, we report that an evolutionarily conserved tandem Tudor domain (TTD) in the BAH Domain And Coiled-Coil Containing 1 (BAHCC1) protein (BAHCC1_TTD) selectively reads H4K20me1 for facilitating the replication origin activation and efficient DNA replication. Our integrated biochemical, structural, genomic and cellular analyses demonstrated that BAHCC1_TTD preferentially recognizes H4K20me1 to promote the recruitment of BAHCC1 and its interacting partners, notably the Mini-chromosome Maintenance (MCM) complex, to the replication origin sites. Combined actions of the H4K20me1-reading BAHCC1 and the H4K20me2-reading Origin Recognition Complex (ORC) ensure optimal genomic loading of MCM complexes for facilitating DNA replication. Depletion of BAHCC1, or disruption of the BAHCC1_TTD:H4K20me1 interaction, reduces levels of H4K20me1 and MCM loading, leading to defects in replication and cell cycle progression. Together, this study identifies BAHCC1_TTD as a conserved effector transducing the H4K20me1 signals to the recruitment of MCM complexes for facilitating efficient DNA replication.
2025-06-03 | GSE293272 | GEO