Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis
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ABSTRACT:
PROVIDER: PRJNA779072 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16892900_1.fastq.gz | Fastqsanger.gz | |||
| SRR16892900_2.fastq.gz | Fastqsanger.gz | |||
| SRR16892901_1.fastq.gz | Fastqsanger.gz | |||
| SRR16892901_2.fastq.gz | Fastqsanger.gz | |||
| SRR16892902_1.fastq.gz | Fastqsanger.gz |
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Project description:Histone chaperones and chromatin remodelers control nucleosome dynamics, essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display an exclusive preference for the H3.3-depositing HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefer the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation, and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated H3.3-H4 deposition via the HIRA pathway for proper epigenetic regulation of the genome.
2022-10-31 | PXD035578 | Pride
Project description:Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.
2022-10-27 | GSE188493 | GEO
Project description:A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between histone H3–H4 chaperones in the histone chaperone network. We identify several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3–H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.
2023-03-02 | PXD034888 | Pride
Project description:A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between histone H3–H4 chaperones in the histone chaperone network. We identify several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3–H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.
2023-03-02 | PXD038263 | Pride
Project description:Histones are the protein components of the basic unit of chromatin, the core particle of the nucleosome. They play a central role in defining chromatin states associated with distinct cell fates and are classified into replicative and non-replicative/replacement histone variants. While the latter do not exhibit S phase regulation in their expression, the replicative histone variants show a major peak in expression early during S phase to support chromatin assembly during replication of the genome. Their expression is tightly regulated during the cell-cycle both transcriptionally and post-transcriptionally and involves a number of actors. During replication in human cells, two main chaperones ensure the deposition of H3-H4 onto DNA: Chromatin assembly factor 1 (CAF-1) and Anti-silencing factor 1 (ASF1). Interestingly, on the one hand, ASF1 binds the newly synthesized replicative histones H3.1/H3.2-H4 to hand them off to the downstream chaperone, CAF-1, for deposition onto the duplicated DNA strands in a DNA synthesis-coupled (DSC) manner. On the other hand, ASF1 also promotes the recycling of parental histones during replication. In addition, ASF1 binds the non-replicative variant H3.3 and hands it off to the downstream chaperone Histone regulator A (HIRA) for deposition of H3.3 in a DNA synthesis independent (DSI) manner. Finally, in human cells, ASF1 but not CAF-1, also provides a buffering system for histone excess generated in response to stalled replication, indicating yet another role for ASF1 in regulating the flow of replicative histones in higher eukaryotes. However, to date, roles of these chaperones in histone RNA metabolism in mammals had remained unexplored. This is particularly interesting to consider given that in budding yeast, where there are no distinct replicative and non-replicative H3 variants, the single ASF1 ortholog participates in activating transcription of histone genes in S phase and transcriptional repression outside S phase in combination with Hir1, the budding yeast counterpart of HIRA. We thus decided to explore how the key histone chaperones involved in DNA synthesis-coupled chromatin assembly could contribute to the critical regulation of expression of replicative histone genes in human cells during S phase. From total RNA extracted from asynchronous and synchronized human cells, we performed RNA-seq and found that most of the annotated replicative histone genes decreased in expression upon ASF1 depletion by siRNA during S phase compared to the control condition with siRNA againt GFP. However, by 4sU-labeled RNA-seq we detected an increase in newly synthesized replicative histone transcripts. These findings indicate that the decrease in expression of replicative histone genes in ASF1-depleted cells cannot be due to a decrease at the level of transcription. We then inspected closely the sequences at the 3’ end of the replicative histone transcripts in our RNA-seq data and detected a defect of their 3’ processing. Thus, we propose that in mammals ASF1 plays a role in the unique regulation of replicative histone RNA metabolism.
2023-11-29 | E-MTAB-12501 | biostudies-arrayexpress
Project description:Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to specifically recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive posttranslational histone modifications. H3.Y mutational gain-of-function analyses screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core region and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, ChIP-seq experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin.
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Project description:The histone acetyltransferase Sas2 is part of the SAS-I complex and acetylates lysine 16 of histone H4 (H4 K16Ac) in the genome of Saccharomyces cerevisiae. Sas2-mediated H4 K16Ac is strongest over the coding region of genes with low expression. However, it is unclear how Sas2-mediated acetylation is incorporated into chromatin. Our previous work has shown physical interactions of SAS with the histone chaperones CAF-I and Asf1, suggesting a link between SAS-I mediated acetylation and chromatin assembly. Here, we find that Sas2-dependent H4 K16Ac in bulk histones requires passage of the cells through the S-phase of the cell cycle, and the rate of increase in H4 K16Ac depends on both CAF-I and Asf1, whereas steady-state levels and genome-wide distribution of H4 K16Ac shows only mild changes in their absence. Furthermore, H4 K16Ac is deposited in chromatin at genes upon repression, and this deposition requires the histone chaperone Spt6, but not CAF-I, Asf1, HIR or Rtt106. Altogether, our data indicate that Spt6 controls H4 K16Ac levels by incorporating K16-unacetylated H4 in strongly transcribed genes. Upon repression, Spt6 association is decreased, resulting in less deposition of K16-unacetylated and therefore in a concomitant increase of H4 K16Ac that is recycled during transcription.
2015-09-14 | E-MTAB-2638 | biostudies-arrayexpress
Project description:We developed a new sequencing assay to track the de novo deposition of the histone H3 variants H3.1 and H3.3 during S phase. We use cells stably expressing H3.1-SNAP or H3.3-SNAP, and synchronize them in G1/S by double-thymidine block. The SNAP-tag enables to discriminate newly synthesized histones from preexisting ones, via a quench-chase-capture strategy. We applied this strategy to isolate new H3.1 and H3.3 after releasing cells into S phase, and probed their distribution by MNase digestion and sequencing. We could thus characterize H3.1 and H3.3 dynamics from early to mid S phase at genome-wide resolution. We further applied our method to investigate the consequences of perturbations upon deletion of the H3.3 chaperone HIRA. We used HIRA knockout and control cells, and compared H3.1 and H3.3 distribution to early replication patterns by EdU labeling and sequencing of nascent DNA.
2022-03-31 | E-MTAB-10619 | biostudies-arrayexpress
Project description:Promyelocytic Leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. Here, by using specific siRNAs and protein Affimers, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. In addition, we demonstrate that HIRA localization in the nuclear bodies is intimately linked to the presence of a soluble pool of H3.3-H4 dimers inside PML NBs, that is not found in cancer cells. Transcription inhibition prevents HIRA accumulation in PML NBs underscoring the importance of transcriptional activity to drive HIRA through PML NBs. Finally, in the context of inflammatory responses, HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of interferon-stimulated genes (ISGs), well beyond the peak of transcription. We thus propose that HIRA partitioning in PML NBs is essential to regulate H3.3 deposition on transcriptionally active regions.
2023-05-25 | GSE233298 | GEO
Project description:Promyelocytic Leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. Here, by using specific siRNAs and protein Affimers, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. In addition, we demonstrate that HIRA localization in the nuclear bodies is intimately linked to the presence of a soluble pool of H3.3-H4 dimers inside PML NBs, that is not found in cancer cells. Transcription inhibition prevents HIRA accumulation in PML NBs underscoring the importance of transcriptional activity to drive HIRA through PML NBs. Finally, in the context of inflammatory responses, HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of interferon-stimulated genes (ISGs), well beyond the peak of transcription. We thus propose that HIRA partitioning in PML NBs is essential to regulate H3.3 deposition on transcriptionally active regions.
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