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.
Highlights
IntroductionGenomic DNA is associated with proteins to form chromatin. The basic sub-unit of chromatin 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 DNA1
In eukaryotes, genomic DNA is associated with proteins to form chromatin
In order to determine that exogenous histones were not competing with endogenous histones to form a hybrid complex, histones H3 and H4 containing a FLAG-tag epitope were individually prepared as well as a complex of H3 and FLAG-tagged H4, and trace amounts of exogenous histones were incorporated into syncytial plasmodium fragments of Physarum at S-phase onset
Summary
Genomic DNA is associated with proteins to form chromatin. The basic sub-unit of chromatin 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 DNA1. 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]. The high conservation of the deposition-related di-acetylation of H4 suggested that the H4 amino-terminal tail domain was required for replication. The incorporation of trace amounts of exogenous histones within naturally synchronized Physarum cells proved powerful in overruling the experimental bias[17] As this approach distinguishes the histone nuclear import and chromatin assembly, it is possible to determine the requirements for each process. Incorporation of histone dimers lacking the terminal tail domains in Physarum did not lead to a nuclear import defect, suggesting that the histone complex presented features that enabled their transportation into the nucleus[17]. Biochemical analyses of the factors involved in the nuclear import of newly synthesized histones exhibited a high degree of conservation for this pathway between eukaryotes[21]
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