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The yeast genome is globally accessible in living cells [OxfordNanopore]

ABSTRACT: Eukaryotic genomes are packaged into chromatin, which is composed of condensed filaments of regularly spaced nucleosomes, resembling beads on a string. The nucleosome contains ~147 bp of DNA wrapped almost twice around a central core histone octamer. The packaging of DNA into chromatin represents a challenge to transcription factors and other proteins requiring access to their binding sites. Consequently, control of DNA accessibility is thought to play a key role in gene regulation. Here, we measure DNA accessibility genome-wide in living budding yeast cells by inducible expression of DNA methyltransferases. We find that the yeast genome is globally accessible to the methylase in living cells, unlike in nuclei, where DNA accessibility is severely limited. Gene bodies are methylated at only slightly slower rates than promoters, indicating that yeast chromatin is highly dynamic in vivo. In contrast, centromeres are strongly protected in vivo. Global shifts in nucleosome positions occur in cells as they are depleted of RSC, or ISW1 and CHD1, indicating that global nucleosome dynamics are at least partly attributable to ATP-dependent chromatin remodelers. We propose that virtually all yeast chromatin is in a state of continuous flux in living cells, but static in nuclei, suggesting that chromatin packaging is not generally repressive.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE230306 | GEO | 2024/03/22

REPOSITORIES: GEO

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The yeast genome is globally accessible in living cells [Illumina]

Project description:Eukaryotic genomes are packaged into chromatin, which is composed of condensed filaments of regularly spaced nucleosomes, resembling beads on a string. The nucleosome contains ~147 bp of DNA wrapped almost twice around a central core histone octamer. The packaging of DNA into chromatin represents a challenge to transcription factors and other proteins requiring access to their binding sites. Consequently, control of DNA accessibility is thought to play a key role in gene regulation. Here, we measure DNA accessibility genome-wide in living budding yeast cells by inducible expression of DNA methyltransferases. We find that the yeast genome is globally accessible to the methylase in living cells, unlike in nuclei, where DNA accessibility is severely limited. Gene bodies are methylated at only slightly slower rates than promoters, indicating that yeast chromatin is highly dynamic in vivo. In contrast, centromeres are strongly protected in vivo. Global shifts in nucleosome positions occur in cells as they are depleted of RSC, or ISW1 and CHD1, indicating that global nucleosome dynamics are at least partly attributable to ATP-dependent chromatin remodelers. We propose that virtually all yeast chromatin is in a state of continuous flux in living cells, but static in nuclei, suggesting that chromatin packaging is not generally repressive.

2024-03-22 | GSE229797 | GEO

DNase02

Project description:In eukaryotes, DNA is packaged into chromatin. It has become evident that the degree of packaging is not uniform along the genome. More compact chromatin greatly hinder access of transcription factors or other DNA-binding or modifying proteins, and DNA accessibility is an important yet poorly studied layer of regulation. The aim of the study was to map DNA accessibility in chromatin of Arabidopsis leaf nuclei using DNaseI sensitivity as a proxy for accessibility. Chromatin was digested with DnaseI and genomic fragments of >17kb were hybridized to AGRONOMICS1 tiling arrays.

2013-05-01 | E-TABM-1219 | biostudies-arrayexpress

DNA accessibility is not the primary determinant of chromatin-mediated gene regulation (yeast III)

Project description:DNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at all genomic AluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, and indicating that DNA accessibility is primarily determined by nucleosome spacing. Remarkably, AluI sites in inactive mouse promoters are accessible in some cells. Furthermore, euchromatin and heterochromatin have very similar accessibilities. Cell-to-cell heterogeneity in DNA accessibility has implications for chromatin models of gene regulation: a transcription factor binding site is blocked in some cells, but not in all cells, guaranteeing neither activation nor repression.

2019-08-27 | GSE120956 | GEO

DNA accessibility is not the primary determinant of chromatin-mediated gene regulation (yeast II)

2019-08-27 | GSE120955 | GEO

DNA accessibility is not the primary determinant of chromatin-mediated gene regulation (mouse)

2019-08-27 | GSE115691 | GEO

In vivo Analysis Reveals that ATP-hydrolysis Couples Remodeling to SWI/SNF Release from Chromatin.

Project description:ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.

2022-01-20 | PXD025474 | Pride

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Project description:Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ~147 bp of DNA wrapped ~1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ~160 bp, and then converts it to a core particle, containing ~147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these "proto-chromatosomes" are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

2015-09-23 | GSE65889 | GEO

Functional DNA methylation is accompanied by chromatin accessibility [methylation]

Project description:Analysis of nucleosome positioning and chromatin state by using CpG methyltransferase M.SssI to methylate nuclei. Unmethylated regions that gain methylation (low to high beta value) are known to be accessible and nucleosome depleted. Method used to study changes after epigenetic drug treatments identified that majority of demethylation events are not accompanied by chromatin accessibility changes. Intact nuclei are harvested from cells and treated with M.SssI. DNA is then extracted, bisulfite converted and run on an Infinium methylation array, along with a no-enzyme control. Background subtracted beta values (listed below) are used to determine regions that have gained methylation on enzyme treatment compared to the control - and these are used to infer chromatin state

2013-03-08 | E-GEOD-43851 | biostudies-arrayexpress

Structure of transcription preinitiation complex engaged with the +1 nucleosome

Project description:The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles in chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is positioned downstream of the core promoter. Here we present the cryo-EM structure of the yeast PIC bound to promoter DNA and the +1 nucleosome at a resolution of 3.4–3.9 Å. The general transcription factor TFIIH forms four contacts with the nucleosome, indicating how PIC assembly can be assisted by the +1 nucleosome. The TFIIH subunit Ssl2 (XPB in human TFIIH) forms a wedge between promoter DNA and the nucleosome, stabilizing two turns of DNA that are detached from the nucleosome. During subsequent scanning for the transcription start site (TSS), three additional turns of DNA can be detached before the partially unraveled nucleosome may counteract further scanning, explaining why TSSs are located at a distance of ~60 bp from the dyad of the +1 nucleosome in yeast.

2023-03-11 | PXD029840 | Pride

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin

Project description:Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

2018-12-04 | GSE100401 | GEO

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