The single-molecule accessibility landscape of newly replicated mammalian chromatin
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE245558 | GEO | 2024/11/12
REPOSITORIES: GEO
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Project description:Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediates these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility, the insulator function of CTCF is nevertheless impaired resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function.
2024-03-13 | PXD042945 | Pride
Project description:The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription. Here we systematically integrated multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, CRISPR-Cas9 survival dropout screening, time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewired genome-wide chromatin accessibility. Increased accessible chromatin regions were largely located adjacent to CTCF-binding sites at promoter regions and insulator sites and were associated with enhanced transcription of nearby genes. In addition, we used CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners in regulating downstream gene expression. We successfully identified 40 candidates, including multiple established partners (i.e., MYC) supported by all layers of evidence. Interestingly, many CTCF co-regulators (e.g., YY1, ZBTB7A) that have evident alterations of respective downstream gene expression do not show changes at their expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription. This study highlights CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation
2021-07-04 | PXD026484 | Pride
Project description:Replication of the eukaryotic genome occurs in the context of chromatin, a nucleoprotein packaging state consisting of repeating nucleosomes. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture that occur during genomic replication. Here, we sought to directly measure a key aspect of chromatin structure dynamics during replication â how rapidly nucleosome positions are established on the newly-replicated daughter genomes. By isolating newly-synthesized DNA marked with the nucleotide analogue EdU, we characterize nucleosome positions on both daughter genomes of budding yeast during a time course of chromatin maturation. We find that nucleosomes rapidly adopt their mid log positions at highly-transcribed genes, and that this process was impaired upon treatment with the transcription inhibitor thiolutin, consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in the Hir1Î background reveal a role for HIR in nucleosome spacing. Using strand-specific EdU libraries, we characterize nucleosome positions on the leading and lagging strand daughter genomes, uncovering differences in chromatin maturation dynamics between the two daughter genomes at hundreds of genes. Our data define the maturation dynamics of newly-replicated chromatin, and support a role for transcription in sculpting the chromatin template. We have mapped changes in nucleosome positions on newly replicated DNA in a timecourse after genome replication. We have used Micrococcal Nuclease footprinting of cross linked chromatin to determine nucleosome positions and EdU (ethylene deoxy uridine) to mark nascent DNA strands. EdU incorporated into nascent DNA strands was biotinylated with Click chemistry and nascent DNA strand fragments were subsequently isolated using Streptavidin coated magnetic beads.
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Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to Nascent Chromatin Capture, we provide time resolved binding kinetics for thousands of proteins behind replisomes of mid-S replicated regions. This shows that most proteins regain access within the first 15 minutes after the passage of the fork. In contrast, 25% of the identified proteins do not, and this delay cannot be inferred from their known function, physicochemical properties, or nuclear abundance. Here, we provide data where we impaired the H3K27me3 restoration to analyse its impact into chromatin restoration. To this aim, we used the EPZ-6438 inhibitor, which blocks the histone methyltransferase EZH2. Two time-points are analysed by NCC along the cell cycle in late replicated regions: nascent chromatin, just after the passage of the fork, and a sample 16 hours later, in late G1 phase. Our data support that H3K27me3 have an important role in chromatin restoration. Data available in this entry are related with the supplementary table 6 from our manuscript.
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Project description:Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative MS-based proteomics coupled to Nascent Chromatin Capture, we provide time resolved binding kinetics for thousands of proteins behind replisomes of mid-S replicated regions. This shows that most proteins regain access within the first 15 minutes after the passage of the fork. In contrast, 25% of the identified proteins do not, and this delay cannot be inferred from their known function, physicochemical properties, or nuclear abundance. A sample treated with nocodazole to block the cell cycle progression. The comparison between a normal cell cycle progression and a nocodazole treated sample shows that the reassociation onto chromatin of proteins with a delayed restoration can be regulated by the cell cycle progression and/or the post-replication time. In addition, we provide evidence that DNA replication not only disrupts but also promotes recruitment of transcription factors and chromatin remodellers, providing a significant advance in understanding how DNA replication could contribute to programmed changes of cell memory. Data available in this entry are related with the supplementary table 5 from our manuscript.
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Project description:In the nucleus of the eukaryote cell, the genetic material is organized in the form of chromatin where DNA associates with nucleosomes, formed by an octamer of histone proteins, to provide the correct environment to regulate gene expression, maintain cellular identity and avoid disease. Despite the important role of chromatin organization in cellular function, in each cell division chromatin is totally disorganized. Nucleosomes are evicted ahead of the replication fork and will have to be recycled together with newly synthetized histones to maintain nucleosome density in the two daughter strands. This produce changes in chromatin accessibility and a dilution in epigenetic information. To date, it is not clear how these chromatin and epigenetic changes can affect gene expression. Here, we have used the cutting-edge technology ChOR-seq (Chromatin Occupancy after Replication and sequencing) and ChrRNA-seq (Chromatin-associated RNA-seq) to investigate the distribution and abundance of the RNA Polymerase II (RNAPII) in newly replicated chromatin and nascent RNA levels in human cells.
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Project description:In the nucleus of the eukaryote cell, the genetic material is organized in the form of chromatin where DNA associates with nucleosomes, formed by an octamer of histone proteins, to provide the correct environment to regulate gene expression, maintain cellular identity and avoid disease. Despite the important role of chromatin organization in cellular function, in each cell division chromatin is totally disorganized. Nucleosomes are evicted ahead of the replication fork and will have to be recycled together with newly synthetized histones to maintain nucleosome density in the two daughter strands. This produce changes in chromatin accessibility and a dilution in epigenetic information. To date, it is not clear how these chromatin and epigenetic changes can affect gene expression. Here, we have used the cutting-edge technology ChOR-seq (Chromatin Occupancy after Replication and sequencing) and ChrRNA-seq (Chromatin-associated RNA-seq) to investigate the distribution and abundance of the RNA Polymerase II (RNAPII) in newly replicated chromatin and nascent RNA levels in human cells.
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Project description:In the nucleus of the eukaryote cell, the genetic material is organized in the form of chromatin where DNA associates with nucleosomes, formed by an octamer of histone proteins, to provide the correct environment to regulate gene expression, maintain cellular identity and avoid disease. Despite the important role of chromatin organization in cellular function, in each cell division chromatin is totally disorganized. Nucleosomes are evicted ahead of the replication fork and will have to be recycled together with newly synthetized histones to maintain nucleosome density in the two daughter strands. This produce changes in chromatin accessibility and a dilution in epigenetic information. To date, it is not clear how these chromatin and epigenetic changes can affect gene expression. Here, we have used the cutting-edge technology ChOR-seq (Chromatin Occupancy after Replication and sequencing) and ChrRNA-seq (Chromatin-associated RNA-seq) to investigate the distribution and abundance of the RNA Polymerase II (RNAPII) in newly replicated chromatin and nascent RNA levels in human cells.
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