Abstract
BackgroundAnti-Silencing Factor 1 (ASF1) is a conserved H3–H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosome assembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3–H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatin assembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. Here, we have investigated the implication of ASF1 in these two distinct assembly processes.ResultsWe show that depletion of the maternal pool of ASF1 with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, we show that asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, we found that ASF1 is required for the formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, we show that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility.ConclusionsWe conclude that ASF1 is critically required to load H3.3–H4 dimers on the HIRA complex prior to histone deposition on paternal DNA. This separation of tasks could optimize the rapid assembly of paternal chromatin within the gigantic volume of the egg cell. In contrast, ASF1 is surprisingly dispensable for the amplification of cleavage nuclei, although chromatin integrity is likely compromised in KD embryos.
Highlights
Anti-Silencing Factor 1 (ASF1) is a conserved H3–H4 histone chaperone involved in both ReplicationCoupled and Replication-Independent (RI) nucleosome assembly pathways
To address the function of maternal ASF1, we chose to knock-down asf1 expression in adult female germ cells using transgenic small hairpin RNAs whose expression can be induced with germline-specific GAL4 drivers [31]
We turned to another small hairpin RNA (shRNA) (GL00171), which targets the predicted 5′untranslated region (UTR) of asf1 (Fig. 1a)
Summary
Anti-Silencing Factor 1 (ASF1) is a conserved H3–H4 histone chaperone involved in both ReplicationCoupled and Replication-Independent (RI) nucleosome assembly pathways. Discovered in the budding yeast, Anti-Silencing Factor 1 (ASF1) is a small, conserved histone chaperone of the H3/H4 family that is involved in a variety of chromatinrelated functions, such as nucleosome assembly and disassembly, chromatin remodeling, gene silencing or DNA damage checkpoint [6,7,8,9]. This diversity of functions reflects the implication of ASF1 in both ReplicationCoupled (RC) and Replication-Independent (RI) histone deposition pathways. ASF1 and HIRA cooperate for the transcriptional silencing of heterochromatin regions in fission yeast [22] and the formation of senescence-associated heterochromatin foci in human cells [13]
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