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The absence of functional CAF-1 results in deficiencies in H3.3 levels at transcribed loci in Arabidopsis

ABSTRACT: Eukaryotic organisms organize their genetic material in chromatin rather than naked DNA. Efficient chromatin assembly is essential for all DNA-templated processes, including replication, transcription, and DNA repair. Our research focused on two major histone H3 deposition complexes: Chromatin Assembly Factor (CAF-1), which deposits canonical H3 on naked DNA immediately after replication during the S-phase in yeast and metazoans, and the Histone Regulator A (HIRA) complex, which is thought to incorporate the H3.3 variant in a replication-independent manner. By combining biochemical, genetic, genomic, and proteomic data, we revealed that CAF-1 activity might be associated with the maintenance of the histone variant H3.3 in Arabidopsis chromatin. Moreover, this CAF-1's potential function in H3.3 homeostasis may be particularly significant in the transcribed regions of the genome.

ORGANISM(S): Arabidopsis Thaliana

SUBMITTER: Israel Ausin

PROVIDER: PXD081428 | iProX | Tue Jul 21 00:00:00 BST 2026

REPOSITORIES: iProX

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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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The absence of functional CAF-1 results in deficiencies in H3.3 levels at transcribed loci in Arabidopsis

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