Histones H3 and H4 require their relevant amino-tails for efficient nuclear import and replication-coupled chromatin assembly in vivo

Abstract : Concomitant chromatin assembly and DNA duplication is essential for cell survival and genome integrity, and requires newly synthesized histones. Although the N-terminal domains of newly synthesized H3 and H4 present critical functions, their requirement for replication-coupled chromatin assembly is controversial. Using the unique capability of the spontaneous internalization of exogenous proteins in Physarum, we showed that H3 and H4 N-tails present critical functions in nuclear import during the S-phase, but are dispensable for assembly into nucleosomes. However, our data revealed that chromatin assembly in the S-phase of complexes presenting ectopic N-terminal domains occurs by a replication-independent mechanism. We found that replication-dependent chromatin assembly requires an H3/H4 complex with the relevant N-tail domains, suggesting a concomitant recognition of the two histone domains by histone chaperones. In eukaryotes, genomic DNA is associated with proteins to form chromatin. The basic sub-unit of chroma-tin is the nucleosome composed of a central tetramer of H3/H4, flanked by two heterodimers of H2A/H2B, and the octamer is wrapped by about two superhelical turns of DNA 1. The core histones are composed of two distinct domains; the fold domain involved in the histone-histone interaction with the nucleosome and the amino-tail domain that extends outside the nucleosome 2, 3. The histone tail domains have been shown to be post-translationally modified and these modifications are generally believed to be involved in chromatin activity regulation 4. During the S-phase of the cell cycle, the genome replicates and, in conjunction with DNA synthesis, chro-matin is assembled 5. The doubling of the genetic material associated with replication requires parental histone dilution and the synthesis of new histones to compact DNA within the nucleus. Using a pulse labeling strategy for studying newly synthesized histones revealed a conserved di-acetylation on lysines 5 and 12 of histone H4 (corresponding to 4 and 11 in Tetrahymena) related to chromatin deposition 6. This high conservation of the deposition-related di-acetylation of H4 suggested that histone acetyltransferase (HAT) is also highly conserved. In contrast, the newly synthesized H3 acetylation pattern in relation to replication presented a weaker degree of conservation. In the protozoan Tetrahymena, the partitioning of chromatin activities between two distinct nuclei, i.e. a macronucleus that transcribes and replicates and a micronucleus that only replicates, enabled HAT activities to be distinguished 7. Preparation of extracts from micronuclei and cytoplasm revealed that the deposition-related di-acetylation of H4 was catalyzed by a type B histone acetyltransferase, which does not acetylate histone in the chromatin form 8. This type B HAT was first isolated from yeast cytoplasmic extracts and corresponds to a two-subunit holoenzyme with Hat1p as the catalytic subunit and Hat2p 9. Consistently with the conservation of the deposition-related di-acetylation of H4, Human HAT1 has been identified and presents a high degree of conservation 10. Like the yeast enzyme, Human HAT1 is composed of two sub-units, Hat1 and RbA-p46. However, the enzyme was shown to have a nuclear localization in the vicinity of replication forks during the S-phase 10, 11. Furthermore, the chromatin assembly complex, which is composed of the three subunits of CAF-1 (p150, p60 and RbA-p48) and H3/H4 and which promotes replication-dependent chromatin assembly, exhibited acetylation of
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Aïda Ejlassi, Vanessa Menil-Philippot, Angélique Galvani, Christophe Thiriet. Histones H3 and H4 require their relevant amino-tails for efficient nuclear import and replication-coupled chromatin assembly in vivo. Scientific Reports, Nature Publishing Group, 2017, 7 (1), pp.3050 ⟨10.1038/s41598-017-03218-6⟩. ⟨inserm-01856271⟩

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