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A Basic Domain in the Histone H2B N-terminal Tail is Important for Nucleosome Assembly by FACT

ABSTRACT: Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE80831 | GEO | 2016/08/10

SECONDARY ACCESSION(S): PRJNA320099

REPOSITORIES: GEO

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A Basic Domain in the Histone H2B N-terminal Tail is Important for Nucleosome Assembly by FACT

Project description:Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT. 6 samples, 2 inputs and 4 ChIP samples for histone H2B (2 for wild-type and 2 for an H2B â30-37 mutant)

2016-08-10 | E-GEOD-80831 | biostudies-arrayexpress

Molecular basis of RanGTP-activated release of Histones H2A-H2B from Importin-9

Project description:Imp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP is insufficient to release H2A-H2B. The resulting stable RanGTP•Imp9•H2A-H2B complex gains nucleosome assembly activity with H2A-H2B able to be deposited into an assembling nucleosome in vitro. Using hydrogen-deuterium exchange coupled with mass spectrometry (HDX), we show that Imp9 stabilizes H2A-H2B beyond the direct binding site, like other histone chaperones. HDX also shows that binding of RanGTP releases H2A-H2B contacts at Imp9 HEAT repeats 4-5, but not 18-19. DNA- and histone-binding surfaces of H2A-H2B are exposed in the ternary complex, facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. Imp9 thus provides a connection between the nuclear import of H2A-H2B and its deposition into chromatin.

2023-06-02 | PXD037571 | Pride

Recognition of inherently unstable H2A-nucleosome by Swc2 is a major determinant for unidirectional H2A.Z exchange

Project description:The site-specific chromatin incorporation of eukaryotic histone variant H2A.Z is driven by the multi-component chromatin remodeling complex SWR1/SRCAP/ p400. The budding yeast SWR1 complex replaces the H2A-H2B dimer in the canonical nucleosome with the H2A.Z-H2B dimer, but the mechanism governing the directionality of H2A-to-H2A.Z exchange remains elusive. Here, we use single-molecule force spectroscopy to dissect the disassembly/ reassembly of H2A-nucleosome and H2A.Z-nucleosome. We find that the N-terminal 1-135 residues of yeast SWR1-complex-protein-2 (previously termed Swc2-Z) facilitate the disassembly of nucleosomes containing H2A but not H2A.Z. The Swc2-mediated nucleosome disassembly/reassembly requires the inherently unstable H2A-nucleosome, whose instability is conferred by three H2A α2-helix residues Gly47, Pro49 and Ile63 as they selectively weaken the structural rigidity of H2A-H2B dimer. It also requires Swc2-ZN (residues 1-37) that directly anchors to H2A-nucleosome and functions in the SWR1-catalyzed H2A.Z replacement in vitro and yeast H2A.Z deposition in vivo. Our findings providecrucial insights into how SWR1 complex discriminates between the H2A-nucleosome and H2A.Z-nucleosome, establishing a simple paradigm for the governace of unidirectional H2A.Z exchange.

2021-04-12 | GSE171844 | GEO

The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A

Project description:Eukaryotic chromatin structure is highly conserved, with the canonical histone proteins revealing only small sequence changes across species. Yet, all vertebrates exhibit three much larger histone H2A variants, macroH2A. A distinctive feature of these atypical histones is the globular macrodomain module, which can bind metabolites and is connected to the histone fold through a flexible linker. MacroH2A variants impact heterochromatin organization, transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how these large H2A variants are incorporated into chromatin and the identity of any chaperones required for histone deposition have remained elusive. Here, we developed a split-GFP-based cellular readout for histone incorporation and conducted a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We identified and validated the histone chaperone ANP32B as a regulator of macroH2A chromatin deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetraso me. In cells, depletion of ANP32B in cells strongly affects global macroH2A deposition, revealing ANP32B as a macroH2A chaperone. Our study highlights the power of haploid cell functional genomics coupled with cellular imaging to identify factors that are required for chromatin plasticity and diversity.

2023-11-07 | GSE241387 | GEO

Recycling of parental histones preserves the epigenetic landscape during embryonic development (STRIPE-seq)

Project description:Epigenetic inheritance during DNA replication requires an orchestrated assembly of nucleosomes from parental and newly synthesized histones. We analyzed Drosophila HisC mutant embryos harboring a deletion of all canonical histone genes, in which nucleosome assembly relies on parental histones from cell cycle 14 onwards. Lack of new histone synthesis and parental histone recycling leads to more accessible chromatin and reduced nucleosome occupancy. This leads to upregulated and spurious transcription, whereas the control of the developmental transcriptional program is partially maintained. Importantly, the genomic positions of modified parental H2A, H2B, and H3 are restored during DNA replication. Therefore, the results suggest that parental histones with active or repressive marks are recycled to preserve the epigenetic landscape during DNA replication in vivo.

2022-12-27 | GSE203477 | GEO

Recycling of parental histones preserves the epigenetic landscape during embryonic development (CUT&Tag)

2022-12-27 | GSE203476 | GEO

Recycling of parental histones preserves the epigenetic landscape during embryonic development (CUTAC)

2022-12-27 | GSE203474 | GEO

Recycling of parental histones preserves the epigenetic landscape during embryonic development (ATAC-seq)

2022-12-27 | GSE203473 | GEO

Recycling of parental histones preserves the epigenetic landscape during embryonic development (RNA-seq)

2022-12-27 | GSE203471 | GEO

Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome

Project description:Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or no H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction. Total nucleosomes from MNase-treated nuclear extracts were fractionated by sequential immunoprecipitation into homotypic H2A/H2A (AA), heterotypic H2A/H2A.Z (AZ), and homotypic H2A.Z/H2A.Z (ZZ) nucleosomes.

2010-10-12 | E-GEOD-24618 | biostudies-arrayexpress

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