Transcription Restart Establishes Chromatin Accessibility after DNA Replication
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PROVIDER: PRJNA528325 | ENA |
REPOSITORIES: ENA
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Project description:DNA replication is highly disruptive to chromatin, leading to eviction of nucleosomes, RNA polymerase and regulatory factors. When and how transcription resumes on DNA following DNA replication is unknown. Here we develop a replication-coupled Assay for Transposase-Accessible Chromatin, repli-ATAC-seq, to investigate active chromatin restoration post-replication in mouse embryonic stem cells. We find nascent chromatin is inaccessible and transcriptionally silent, with accessibility and RNA polymerase occupancy re-appearing within 30 minutes. Chromatin accessibility restores differentially genome-wide, with super enhancers regaining transcription factor occupancy faster than other genomic features. We also identify opportunistic and transient accessible chromatin within gene bodies after replication. Systematic inhibition of transcription shows that transcription restart is required to re-establish active chromatin states genome-wide and resolve opportunistic binding events resulting from DNA replication. Collectively, this establishes a central role for transcription in overcoming the genome-wide chromatin inaccessibility imposed by DNA replication every cell division.
2019-05-21 | GSE128643 | GEO
Project description:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress. In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by M-NM-1-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24M-BM-0C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (M-NM-^Tmec1 M-NM-^Tsml1 and mec1-100 M-NM-^Tsml1), as well as control strains (M-NM-^Tsml1).
2013-01-17 | E-GEOD-43548 | biostudies-arrayexpress
Project description:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress.
2013-01-17 | GSE43548 | GEO
Project description:Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation
| PRJNA661770 | ENA
Project description:Mammalian oocytes have the ability to reset the transcriptional program of differentiated somatic cells into that of totipotent embryos through somatic cell nuclear transfer (SCNT). However, the mechanisms underlying SCNT-mediated reprogramming are largely unknown. To understand the mechanisms governing chromatin reprogramming during SCNT, we profiled DNaseI hypersensitive sites (DHSs) in donor cumulus cells and 1-cell stage SCNT embryos. To our surprise, the chromatin accessibility landscape of the donor cells is drastically changed to recapitulate that of the in vitro fertilization (IVF)-derived zygotes within 12 hours. Interestingly, this DHS reprogramming takes place even in the presence of a DNA replication inhibitor, suggesting that SCNT-mediated DHS reprogramming is independent of DNA replication. Thus, the study not only reveals the rapid and drastic nature of the changes in chromatin accessibility through SCNT, but also provides a DNA replication-independent model for studying cellular reprogramming.
2018-05-14 | GSE110851 | GEO
Project description:Full title: Complex patterns of genome accessibility discriminate sites of PcG repression, H4K16 acetylation and replication initiation Histone modifications have been proposed to regulate gene expression in part by modulating DNA accessibility and higher-order chromatin structure. However, there is limited direct evidence to support structural differences between euchromatic and heterochromatic fibers in the nucleus. To ask how histone modifications relate to chromatin compaction, we measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint). In the Drosophila genome, we find that accessibility to DNA methylase is variable in a manner that relates to the differential distribution of active and repressive histone modifications. Active promoters are highly permissive to M.SssI activity, yet inactive chromosomal domains decorated with H3 lysine 27 trimethylation are least accessible providing in vivo evidence for Polycomb-mediated chromatin compaction. Conversely, DNA accessibility is increased at active chromosomal regions marked with H4 lysine 16 acetylation and at the dosage-compensated male X chromosome suggesting that Drosophila transcriptional dosage compensation is facilitated by more permissive chromatin structure. Interestingly early replicating chromosomal regions and sites of replication initiation show also higher accessibility linking temporal and spatial control of genome duplication to the structural organization of chromatin. In conclusion, using a novel protocol we generated a comprehensive view of DNA accessibility and uncover different levels of chromatin organization, which are delineated by distinct patterns of posttranslational histone modifications and replication. Keywords: cell type comparison, ChIP-chip, MeDIP-footprint, RNA-seq, ChIP-seq MeDIP-footprint and ChIP-chip: ChIP-chip was performed for H3K4me3, H3K36me2, H3K36me3, H3K27me3, and H3K9me2 in Kc cells. We measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint) in Kc and S2 cells. RNA-seq: cDNA from RNA from Drosophila Kc cells was sequenced using Illumina deep sequencing. Reads were mapped and the abundance of all transcripts was determined. ChIP-seq: PSC ChIP from Drosophila Kc cells was sequenced using Illumina deep sequencing in three lanes. Reads were mapped and the binding profile of PSC was determined.
2010-03-16 | E-GEOD-17697 | biostudies-arrayexpress
Project description:Reprogramming of chromatin accessibility in somatic cell nuclear transfer is DNA replication independent
| PRJNA434693 | ENA
Project description:DNA replication fidelity is essential for maintaining genetic stability. Forks arrested at replication fork barriers can be stabilised by the intra-S phase checkpoint, subsequently being rescued by a converging fork, or resuming when the barrier is removed. However, some arrested forks cannot be stabilised and fork convergence cannot rescue in all situations. Thus, cells have developed homologous recombination-dependent mechanisms to restart persistently inactive forks. To understand the dynamics of HR-restart, we visualized in vivo replication dynamics at an S. pombe replication barrier, RTS1, using polymerase usage sequencing and model replication dynamics by Monte Carlo simulation. We confirm that HR-restarted forks synthesise both strands with Pol d and that Pol a is not used significantly on either strand: the lagging strand template remains as a gap that is filled in later. We further demonstrate that HR-restarted forks progress for >30 kb kilobases without maturing to a d/e configuration and can progress through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, demonstrating the stability of the 3' single strand in the context of increased resection.
2020-07-03 | GSE153731 | GEO
Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.
2022-11-02 | PXD034331 | Pride
Project description:T cell receptor (TCR) stimulation of naive CD8+ T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.
| S-EPMC6750808 | biostudies-literature