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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide maps of HMGD1 and H1-bound nucleosomes.

ABSTRACT: Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1, in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. However, the ratio of HMGD1 to H1 is better correlated with chromatin accessibility, gene expression and nucleosome spacing variation than either protein alone suggesting a competitive mechanism between these proteins. Indeed, we show that HMGD1 and H1 compensate each other’s absence by binding reciprocally to chromatin resulting in changes to nucleosome repeat length and distinct gene expression patterns. Collectively our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study thus provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation. ChIP-seq of HMGD1 and Histone H1 bound nucleosomes as well as MNase-seq of total nucleosome in Drosophila S2 cells

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Graham McVicker

PROVIDER: E-GEOD-49526 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide maps of HMGD1 and H1-bound nucleosomes.

Project description:Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1, in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. However, the ratio of HMGD1 to H1 is better correlated with chromatin accessibility, gene expression and nucleosome spacing variation than either protein alone suggesting a competitive mechanism between these proteins. Indeed, we show that HMGD1 and H1 compensate each other’s absence by binding reciprocally to chromatin resulting in changes to nucleosome repeat length and distinct gene expression patterns. Collectively our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study thus provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation.

2013-12-01 | GSE49526 | GEO

Transcription profiling of mouse AtT20 pituitary gland cells following overexpression or down regulation of NSBP1

Project description:Chromatin architectural protein NSBP1/HMGN5 belongs to the family of HMGN proteins which specifically interact with nucleosomes via Nucleosome Binding Domain, unfold chromatin and affect transcription. Mouse NSBP1 is a new and uncharacterized member of HMGN protein family. NSBP1 is a nuclear protein which is localized to euchromatin, binds to linker histone H1 and unfolds chromatin. We analyzed the effect of altered expression levels of mouse NSBP1 protein on global gene expression profile in AtT20 pituitary cells. We found that NSBP1 modulates the fidelity of cellular transcription in the nucleosome binding-dependent manner. Experiment Overall Design: Stable clones overexpressing wild type mouse NSBP1 protein or mutated NSBP1SE protein which does not bind to nucleosomes were generated by retroviral infection of AtT20 cells. siRNA treatment was applied to down regulate NSBP1 in the cells. Total RNA was collected from biological triplicates and analyzed by Affymetrix expression arrays.

2009-06-23 | E-GEOD-16564 | biostudies-arrayexpress

Expression data from AtT20 mouse pituitary gland cells following overexpression or down regulation of NSBP1

2009-06-13 | GSE16564 | GEO

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

Identification and analysis of phosphorylation within Xenopus laevis linker histone H1.0

Project description:As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulators of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moroever, although phosphorylation is a prevalent posttranslational modification (PTM) within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA conformation for six full-length H1s in which the phosphorylated serine residues were replaced by glutamic acid residues.

2022-08-12 | PXD032292 | Pride

Illumina Sequencing data of the influence of Insulator Proteins on chromatin

Project description:The Drosophila insulator-binding proteins (IBPs) dCTCF/Beaf32 mark the physical borders of chromosomal domains involving co-factors that participate in long-range interactions. Chromosomal borders are further enriched in specific histone modifications yet the implication of histone modifiers and nucleosome dynamics remains largely unknown in such context. Here, we show that IBP depletion impairs nucleosome dynamics over genes flanked by their binding sites. Biochemical purification identifies a key histone methyltransferase of H3K36, NSD/dMes-4, as a novel co-factor of IBPs involved in chromatin accessibility, which specifically co-regulates hundreds of genes flanked by Beaf32/dCTCF. dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-mediated H3K36me3, RNA splicing and nucleosome positioning. Our results unveil a model for how IBPs regulate gene expression and nucleosome dynamics through NSD/dMes-4, which may contribute to regulate H3K27me3 spreading. Together, our data suggest a division of labor for how IBPs may dynamically regulate chromatin organization depending on distinct co-factors. Nucleosome profiles of Beaf32-depleted or Wild-Type control Drosophila S2 cells by MNase-Seq (Illumina/TruSeq) for total/bulk nucleosomes or following immunoprecipitation with anti-H3K27me3 antibodies

2014-04-30 | E-GEOD-57166 | biostudies-arrayexpress

MNase titration reveals differences between nucleosome occupancy and chromatin accessibility

Project description:Chromatin accessibility plays a fundamental role in gene regulation. One mechanism to regulate accessibility is nucleosome placement, which is often measured by quantifying protection of DNA from enzymatic digestion. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC, quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both location of nucleosomes and accessibility to MNase in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares behavior of nucleosomes to that of non-histone proteins. We find that enhancers, promoters and other regulatory regions have changes in accessibility that do not correlate with changes in nucleosome occupancy. Moreover, we show that high nucleosome occupancy does not necessarily preclude high accessibility, revealing novel principles of chromatin regulation.

2016-04-18 | GSE78984 | GEO

Crosslinking mass spectrometry of the p300 catalytic core complexed with the nucleosome

Project description:p300 is a histone acetyltransferase that associates with crucial biological processes. p300 acetylates all four histones in the nucleosome, a basic unit of chromatin, and alters chromatin structure and dynamics. In this study, we performed structural and biochemical analysis to understand the nucleosome binding by p300. Crosslinking mass spectrometry suggests that the p300 catalytic core binds to nucleosomes in multiple binding forms to acetylate different histone tails.

2022-06-29 | PXD033804 | JPOST Repository

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

Project description:The nucleosome plays a central role in genome regulation. Traditional methods for mapping nucleosomes depend on the resistance of the nucleosome core to micrococcal nuclease (MNase). However, the lengths of the protected DNA fragments are heterogeneous, limiting the accuracy of nucleosome position information. To resolve this problem, we removed residual linker DNA by simultaneous digestion of yeast chromatin with MNase and exonuclease III (ExoIII). Paired-end sequencing of mono-nucleosomes revealed not only core particles (145-147 bp), but also intermediate particles in which ~8 bp project from one side (154 bp) or both sides (161 bp) of the nucleosome core. We term these particles "pseudo-chromatosomes" because they are present in yeast lacking linker histone. They are also observed after MNase-ExoIII digestion of chromatin reconstituted using recombinant core histones. We propose that the pseudo-chromatosome provides a DNA framework to facilitate H1 binding. Comparison of budding yeast nucleosome sequences obtained using micrococcal nuclease (MNase-seq) and MNase + exonuclease III (ExoIII) (MNase-ExoIII-seq): wild type cells and hho1-null cells. Nucleosome sequences from native chromatin and H1-depleted chromatin from mouse liver. Nucleosome sequences from a plasmid reconstituted into nucleosomes using recombinant yeast histones or native chicken erythrocyte histones.

2015-09-23 | E-GEOD-65889 | biostudies-arrayexpress

Recruitment of HMGN By H3K27ac Modified Nucleosomes to Regulatory Sites Modulates Transcription Factor Binding to Chromatin

Project description:H3K27ac modified nucleosomes are a major epigenetic mark of active chromatin that can be used to identify cell type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that H3K27ac modified nucleosomes recruit the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 to cell-type specific chromatin regulatory regions. We find that HMGNs bind directly to the acetylated nucleosome and that the H3K27ac residue and linker DNA are necessary and sufficient for the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of several transcription factors with chromatin. These experiments identify an additional mechanism whereby the H3K27ac epigenetic mark promotes chromatin decompaction and provide insights into the molecular mechanism whereby HMGN proteins, which bind to chromatin without DNA sequence specificity, modulate cell type specific gene expression.

2020-09-20 | GSE156697 | GEO

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