MNase analysis of linker histone H1 mutant
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PROVIDER: PRJNA451507 | ENA |
REPOSITORIES: ENA
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Project description:MNase-seq and RNA-seq of linker histone H1 mutant
| PRJNA451506 | ENA
Project description:Transcriptiomic analysis of linker histone H1 mutant
| PRJNA451508 | ENA
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
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
Project description:Linker histone H1 plays a key role in chromatin organization and maintenance, however, our knowledge of the regulation of H1 functions by its posttranslational modifications (PTMs) is very limited. In this study, we report on the generation of homogeneously and site-specifically mono- and di-acetylated H1 (H1 Ac) using genetic code expansion. We used these modified histones to identify and comprehensively characterize the acetylation-dependent cellular interactome for linker histone H1 and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Intriguingly, H1 acetylation-specific interactors comprise translational initiation factors and are involved in transcriptional regulation, suggesting that acetylation of H1 may indeed act as a regulator of the linker histone H1 by modulation of protein-protein interactions.
2021-07-09 | PXD025832 | Pride
Project description:There are six histone H1 variant in chicken. 01H1/02H1/03H1/.10H1/H1L and H1R. In those variant we forcused on H1L and H1R those molecule were most similar H1 among six H1 variants. we established linker histone H1L and H1R deleted mutant in chicken B-cell derived DT40 cell and assay gene expression in normal condition in those mutant cells. The detail charactalization of those mutant cell was published in Takami et al., BBRC 268, 501-508 (2000) and Hashimoto et al., DNA repair (in press) Experiment Overall Design: There are six linker histone H1 variant in chicken. Linker histone was thought to be general gene supplesser. We established linker histone H1 variant mutant in chicken B-cell derived DT40 cell, and analyze total protein by 2D-PAGE to find up- or down- regulatd protein. In these mutant, there are some up regulated protein expression and down regulated protein expression (Takami et al., BBRC 268, 501-508 (2000), PubMedID 10679234). Experiment Overall Design: These 2D-PAGE experiment were limited at low density protein region, we use Affymetrix array to search the gene that were regulated specific linker histone H1 variant mutant. Experiment Overall Design: We assayed total gene expression profile in H1L and H1R deleted mutant cells. Experiment Overall Design: all mutant cells were cultured in normal culture condition in RPMI 1640 medium supplemented with 10 µM 2-mercaptoethanol, 10% FCS (Biowest) and 1% chicken serum (Gibco) at 39.5ËC. Total RNA were isolated from exponently growing DT40 cells.
2008-11-08 | E-GEOD-5114 | biostudies-arrayexpress
Project description:Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. While this process is well studied for core histones and many of their PTMs, this is not the case for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the generation of site-specifically mono-ubiquitylated H1.2 via click chemistry and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that the H1 interactome is generally modulated by ubiquitylation and that site-specific ubiquitylation results in overlapping, but distinct interactomes. We further demonstrate that site-specific ubiquitylation of H1 affects the interaction with enzymes relevant for deubiquitylation and deacetylation. We finally show that site-specific ubiquitylation at position K64 impacts H1-dependent chromatosome assembly as well as H1-induced phase separation. In summary, we demonstrate that site-specific ubiquitylation is a general functional regulator for linker histone H1.
2021-07-20 | PXD020686 | Pride
Project description:Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. While this process is well studied for core histones and many of their PTMs, this is not the case for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the generation of site-specifically mono-ubiquitylated H1.2 via click chemistry and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that the H1 interactome is generally modulated by ubiquitylation and that site-specific ubiquitylation results in overlapping, but distinct interactomes. We further demonstrate that site-specific ubiquitylation of H1 affects the interaction with enzymes relevant for deubiquitylation and deacetylation. We finally show that site-specific ubiquitylation at position K64 impacts H1-dependent chromatosome assembly as well as H1-induced phase separation. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.
2021-06-07 | PXD025258 | Pride
Project description:There are six histone H1 variant in chicken. 01H1/02H1/03H1/.10H1/H1L and H1R. In those variant we forcused on H1L and H1R those molecule were most similar H1 among six H1 variants. we established linker histone H1L and H1R deleted mutant in chicken B-cell derived DT40 cell and assay gene expression in normal condition in those mutant cells. The detail charactalization of those mutant cell was published in Takami et al., BBRC 268, 501-508 (2000) and Hashimoto et al., DNA repair (2007) Keywords: gene expression array-based, count
2007-03-21 | GSE5114 | GEO
Project description:In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create an h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.
2021-03-26 | GSE146948 | GEO