CUT&Tag for H2A.Z using mouse heart tissues from control and Znhit1-deficient mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE194162 | GEO | 2022/04/19
REPOSITORIES: GEO
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Project description:A central problem in gene regulation is to discriminate gene regulatory functions of chromatin composition from the one of chromatin-modifying complexes. This question has been virtually unexplored in the context of the H2A.Z histone variant and its incorporation into nucleosomes by the Snf2 Related CREBBP Activator Protein (SRCAP) complex. Here we combine rapid SRCAP depletion, a SRCAP mutant lacking H2A.Z deposition activity, and quantitative genome-wide approaches to dissect how SRCAP and H2A.Z regulate transcription in pluripotent stem cells. We find that SRCAP exhibits essential H2A.Z-independent functions in preventing DNA binding of lineage-specific transcription factors at enhancers and stimulating transcription factor binding at promoters. In contrast, H2A.Z acts mainly as a transcriptional repressor that requires dynamic reloading by SRCAP throughout the cell cycle. Our study clarifies how the SRCAP-H2A.Z axis broadly orchestrates transcription to promote self-renewal, inhibit differentiation, and maintain plasticity of pluripotent stem cells.
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Project description:Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A-H2B dimer with H2A.Z-H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C. The motor module adopts distinct nucleosome-binding modes in the apo (nucleotide-free), ADP-bound, and ADP-BeFx-bound states, suggesting that ATPase-driven movement destabilizes H2A-H2B by unwrapping the entry DNA and pulls H2A-H2B out of nucleosome through the ZNHIT1 subunit. Structure-guided chromatin immunoprecipitation (ChIP) sequencing analysis confirmed the requirement of H2A-contacting ZNHIT1 in maintaining H2A.Z occupancy on the genome. Our study provides structural insights into the mechanism of H2A-H2A.Z exchange mediated by SRCAP-C.
2024-02-10 | GSE249825 | GEO
Project description:A central problem in gene regulation is to discriminate gene regulatory functions of chromatin composition from the one of chromatin-modifying complexes. This question has been virtually unexplored in the context of the H2A.Z histone variant and its incorporation into nucleosomes by the Snf2 Related CREBBP Activator Protein (SRCAP) complex. Here we combine rapid SRCAP depletion, a SRCAP mutant lacking H2A.Z deposition activity, and quantitative genome-wide approaches to dissect how SRCAP and H2A.Z regulate transcription in pluripotent stem cells. We find that SRCAP exhibits essential H2A.Z-independent functions in preventing DNA binding of lineage-specific transcription factors at enhancers and stimulating transcription factor binding at promoters. In contrast, H2A.Z acts mainly as a transcriptional repressor that requires dynamic reloading by SRCAP throughout the cell cycle. Our study clarifies how the SRCAP-H2A.Z axis broadly orchestrates transcription to promote self-renewal, inhibit differentiation, and maintain plasticity of pluripotent stem cells.
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Project description:A central problem in gene regulation is to discriminate gene regulatory functions of chromatin composition from the one of chromatin-modifying complexes. This question has been virtually unexplored in the context of the H2A.Z histone variant and its incorporation into nucleosomes by the Snf2 Related CREBBP Activator Protein (SRCAP) complex. Here we combine rapid SRCAP depletion, a SRCAP mutant lacking H2A.Z deposition activity, and quantitative genome-wide approaches to dissect how SRCAP and H2A.Z regulate transcription in pluripotent stem cells. We find that SRCAP exhibits essential H2A.Z-independent functions in preventing DNA binding of lineage-specific transcription factors at enhancers and stimulating transcription factor binding at promoters. In contrast, H2A.Z acts mainly as a transcriptional repressor that requires dynamic reloading by SRCAP throughout the cell cycle. Our study demonstrates how the SRCAP-H2A.Z axis broadly orchestrates transcription to promote self-renewal, inhibit differentiation, and maintain plasticity of pluripotent stem cells.
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Project description:High levels of the histone variant H2A.Z are characteristic for melanoma progression and correlate with poor prognosis of melanoma patients. Two chaperone complexes are reported to deposit H2A.Z isoforms into chromatin: SRCAP and P400-TIP60. YL1 is a common subunit of both complexes and directly binds to the H2A.Z-H2B dimer. Here, we showed that the knockdown of SRCAP (SRCAP-specific), P400 (P400-TIP60-specific) and YL1 (shared subunit) results in loss of H2A.Z deposition into chromatin in melanoma cells, confirming them as H2A.Z chaperone subunits in this system. Further, we demonstrated that H2A.Z, YL1 and the SRCAP-specific subunit ZNHIT1 co-localize at active promoters of cell cycle-related genes, such as the transcription factor E2F1. Knockdown of YL1, SRCAP and P400 downregulates E2F1 and its targets, resulting in a loss of proliferation and cell cycle arrest in melanoma cells as seen after knockdown of H2A.Z (Vardabasso et al. 2015). Strikingly, besides H2A.Z loss, we observed a dramatic loss of H4 acetylation in melanoma chromatin upon knockdown of H2A.Z chaperone subunits. The majority of genes whose promoters showed a reduction in H4 acetylation were previously bound by H2A.Z, YL1 and ZNHIT1. This is suggestive of a direct link between H2A.Z deposition and H4 acetylation to promote the expression of underlying genes. In agreement, the genes that lost H4 acetylation were enriched for E2F1 target genes. Interestingly, we additionally found that knockdown of YL1 did not only prohibit cell cycle progression, but also induces apoptosis, which is in contrast to H2A.Z knockdown affecting cell cycle only. In addition, YL1 (but not SRCAP or P400) was found to be overexpressed in melanoma and high YL1 levels were a predictor of poor melanoma patient outcome. These findings provide a rationale for targeting the H2A.Z-YL1 interaction as novel epigenetic strategy for melanoma treatment.
2024-05-10 | GSE242227 | GEO
Project description:High levels of the histone variant H2A.Z are characteristic for melanoma progression and correlate with poor prognosis of melanoma patients. Two chaperone complexes are reported to deposit H2A.Z isoforms into chromatin: SRCAP and P400-TIP60. YL1 is a common subunit of both complexes and directly binds to the H2A.Z-H2B dimer. Here, we showed that the knockdown of SRCAP (SRCAP-specific), P400 (P400-TIP60-specific) and YL1 (shared subunit) results in loss of H2A.Z deposition into chromatin in melanoma cells, confirming them as H2A.Z chaperone subunits in this system. Further, we demonstrated that H2A.Z, YL1 and the SRCAP-specific subunit ZNHIT1 co-localize at active promoters of cell cycle-related genes, such as the transcription factor E2F1. Knockdown of YL1, SRCAP and P400 downregulates E2F1 and its targets, resulting in a loss of proliferation and cell cycle arrest in melanoma cells as seen after knockdown of H2A.Z (Vardabasso et al. 2015). Strikingly, besides H2A.Z loss, we observed a dramatic loss of H4 acetylation in melanoma chromatin upon knockdown of H2A.Z chaperone subunits. The majority of genes whose promoters showed a reduction in H4 acetylation were previously bound by H2A.Z, YL1 and ZNHIT1. This is suggestive of a direct link between H2A.Z deposition and H4 acetylation to promote the expression of underlying genes. In agreement, the genes that lost H4 acetylation were enriched for E2F1 target genes. Interestingly, we additionally found that knockdown of YL1 did not only prohibit cell cycle progression, but also induces apoptosis, which is in contrast to H2A.Z knockdown affecting cell cycle only. In addition, YL1 (but not SRCAP or P400) was found to be overexpressed in melanoma and high YL1 levels were a predictor of poor melanoma patient outcome. These findings provide a rationale for targeting the H2A.Z-YL1 interaction as novel epigenetic strategy for melanoma treatment.
2024-05-10 | GSE246121 | GEO
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.
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Project description:Interchanging canonical histone H2A with variant H2A.Z in chromatin complexes is vital for the proper regulation of transcription, DNA damage repair, and centromere maintenance. However, the physical mechanisms underlying functional differences between H2A and H2A.Z complexes are unclear. Human H2A and H2A.Z have a high degree of sequence and structural conservation, with subtle differences occurring in the H2A DNA-binding loops. In this study, we employ hydrogen-deuterium exchange coupled with mass spectrometry and molecular dynamics simulation to investigate the differences in solution behavior between human H2A-H2B and H2A.Z-H2B. We demonstrate that replacing H2A with H2A.Z enhances the dynamics of the histone dimer, whether it is free, complexed with H3-H4, or within the nucleosome. Enhanced dynamics are observed for H2B, suggesting altered interaction with H2A.Z and DNA. Parallel comparisons between H2A-H2B orthologs from humans and frogs show fewer differences in dynamics. Our findings provide mechanistic insights into the function of histone variants and reveal how differences in dynamics may underlie functional differences between structurally similar proteins.
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Project description:Mechanistic basis of gene regulation by SRCAP and H2A.Z (CUT&Tag)
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Project description:Histone proteins, including their variants, play indispensable roles in chromatin structure and function. The specific histone incorporation is the key to epigenetic features and contributes to cell differentiation and development. To understand how each histone is targeted to and incorporated into the specific chromatin region, we used a new method, in which we introduced epitope-tagged histone complexes in permeabilized cells and let them incorporated into the chromatin. The newly incorporated histone complex is easily distinguished from the parental histones by the epitope-tag. We name this as RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay. We analyzed the correlation between the histone incorporation and the chromatin structure, using the H2A-H2B and H2A.Z-H2B complexes. The histone incorporation mainly occurred at less condensed chromatin (open), suggesting that the condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. This led to the genome-wide distribution of H2A and the exclusion of H2A.Z from the closed chromatin.
2021-04-15 | GSE130947 | GEO