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ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

ABSTRACT: BACKGROUND:Anti-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. RESULTS:We 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. CONCLUSIONS:We 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.

SUBMITTER: Horard B

PROVIDER: S-EPMC5946387 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

Horard Béatrice B Sapey-Triomphe Laure L Bonnefoy Emilie E Loppin Benjamin B

Epigenetics & chromatin 20180511 1

<h4>Background</h4>Anti-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 ...[more]

PMID: 29751847

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Project description:Pachytene piRNAs are PIWI-interacting small RNAs abundantly expressed in pachytene spermatocytes and spermatids in adult mouse testes. Both MIWI and MILI-bound pachytene piRNAs have been found enriched in round spermatids. Miwi-null male mice are sterile due to spermiogenic arrest. In C. elegans, sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of offspring derived from piRNA-deficient sperm display a progressive fertility loss after several generations. In mice, it remains unknown whether MIWI-bound pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of offspring derived from MIWI-bound pachytene piRNA-deficient round spermatids also exhibit transgenerational loss of fertility. Here, we report that Miwi KO round spermatids could fertilize both wild type (WT) and Miwi KO oocytes through round spermatid microinjection (ROSI), and produce healthy and fertile offspring despite the aberrant pachytene piRNA profiles in those Miwi KO spermatids. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice. Method: Round spermatids were purified from WT and Miwi KO adult testes using a mini-STA-PUT method[Methods Enzymol 1993; 225:84-113.]，the purity of round spermatids was >90% based on our previous report[ J Biol Chem 2012; 287:25173-25190.]. Small RNA was isolated from round spermatids using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were assessed using the Agilent 2100 Bioanalyzer. Small RNA-Seq was performed on an Ion Proton sequencer (Life Technologies). Libraries were prepared using the Ion Total RNA-Seq Kit v2 (Invitrogen) with biological triplicates for WT and Miwi KO samples. Resutls:Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice.

Dataset Information

ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

Publications

ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

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