BackgroundWe have previously reported a novel O-GlcNAc modification at serine 40 (S40) of H2A (H2AS40Gc). S40-type H2A isoforms susceptible to O-GlcNAcylation are evolutionarily new and restricted to the viviparous animals; however, the biological function of H2AS40Gc is largely unknown. H2A isoforms are consisted of S40 and alanine 40 (A40) type and this residue on H2A is located in the L1 of the globular domain, which is also known as a variable portion that distinguishes between the canonical and non-canonical H2A variants. In this study, by considering the similarity between the S40-type H2A and histone H2A variants, we explored the function of H2AS40Gc in mouse embryonic stem cells (mESCs).ResultsWe found several similarities between the S40-type H2A isoforms and histone H2A variants such H2AZ and H2AX. mRNA of S40-type H2A isoforms (H2A1 N and H2A3) had a poly(A) tail and was produced throughout the cell cycle in contrast to that of A40-type. Importantly, H2AS40Gc level increased owing to chemical-induced DNA damage, similar to phosphorylated H2AX (γH2AX) and acetylated H2AZ (AcH2AZ). H2AS40Gc was accumulated at the restricted area (± 1.5 kb) of DNA damage sites induced by CRISPR/CAS9 system in contrast to accumulation of γH2AX, which was widely scattered. Overexpression of the wild-type (WT) H2A3, but not the S40 to A40 mutation (S40A-mutant), protected the mESC genome against chemical-induced DNA damage. Furthermore, 3 h after the DNA damage treatment, the genome was almost recovered in WT mESCs, whereas the damage advanced further in the S40A-mutant mESCs, suggesting functions of H2AS40Gc in the DNA repair mechanism. Furthermore, the S40A mutant prevented the accumulation of the DNA repair apparatus such as DNA-PKcs and Rad51 at the damage site. Co-immunoprecipitation experiment in WT and S40A-mutant mESCs revealed that H2AS40Gc physiologically bound to AcH2AZ at the initial phase upon DNA damage, followed by binding with γH2AX during the DNA damage repair process.ConclusionsThese data suggest that H2AS40Gc functions to maintain genome integrity through the DNA repair mechanism in association with AcH2AZ and γH2AX.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0157-x) contains supplementary material, which is available to authorized users.

H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.

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