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Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Yasaman Jami-Alahmadi

LAB HEAD: Zhenhui Zhong

PROVIDER: PXD035578 | Pride | 2022-10-31

REPOSITORIES: Pride

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			Action	DRS
	2017-10-10-140min-wb-Israel-Ausin-ASF1a-1-.PNG	Other
	2017-10-10-140min-wb-Israel-Ausin-ASF1a-1-B.raw	Raw
	2017-10-10-140min-wb-Israel-Ausin-ASF1a-2-B-2.PNG	Other
	2017-10-10-140min-wb-Israel-Ausin-ASF1a-2-B.PNG	Other
	2017-10-10-140min-wb-Israel-Ausin-ASF1a-2-B.raw	Raw

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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.

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Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

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