Hayakawa Koji, Hirosawa Mitsuko, Tani Ruiko, Yoneda Chikako, Tanaka Satoshi, Shiota Kunio
Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo, 113-8657, Japan.
Waseda Research Institute for Science and Engineering, Waseda University, Tokyo, 169-8555, Japan.
Epigenetics Chromatin. 2017 Oct 30;10(1):51. doi: 10.1186/s13072-017-0157-x.
We 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).
We 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.
These data suggest that H2AS40Gc functions to maintain genome integrity through the DNA repair mechanism in association with AcH2AZ and γH2AX.
我们之前报道过H2A的丝氨酸40（S40）位点存在一种新的O-连接N-乙酰葡糖胺修饰（H2AS40Gc）。易发生O-连接N-乙酰葡糖胺糖基化的S40型H2A亚型在进化上是新出现的，且仅限于胎生动物；然而，H2AS40Gc的生物学功能在很大程度上尚不清楚。H2A亚型由S40型和丙氨酸40（A40）型组成，H2A上的这个残基位于球状结构域的L1区，该区域也是区分经典和非经典H2A变体的可变部分。在本研究中，通过考虑S40型H2A与组蛋白H2A变体之间的相似性，我们探索了H2AS40Gc在小鼠胚胎干细胞（mESC）中的功能。
我们发现S40型H2A亚型与组蛋白H2A变体如H2AZ和H2AX之间存在一些相似之处。与A40型相比，S40型H2A亚型（H2A1 N和H2A3）的mRNA具有多聚腺苷酸尾，且在整个细胞周期中都有产生。重要的是，与磷酸化的H2AX（γH2AX）和乙酰化的H2AZ（AcH2AZ）类似，化学诱导的DNA损伤会导致H2AS40Gc水平升高。与广泛分布的γH2AX积累不同，H2AS40Gc在CRISPR/CAS9系统诱导的DNA损伤位点的受限区域（±1.5 kb）积累。野生型（WT）H2A3的过表达，但不是S40突变为A40的突变体（S40A突变体），可保护mESC基因组免受化学诱导的DNA损伤。此外，DNA损伤处理3小时后，WT mESC中的基因组几乎恢复，而S40A突变体mESC中的损伤进一步加剧，这表明H2AS40Gc在DNA修复机制中发挥作用。此外，S40A突变体阻止了DNA修复装置如DNA-PKcs和Rad51在损伤位点的积累。WT和S40A突变体mESC中的免疫共沉淀实验表明，H2AS40Gc在DNA损伤初期与AcH2AZ发生生理性结合，随后在DNA损伤修复过程中与γH2AX结合。
这些数据表明，H2AS40Gc通过与AcH2AZ和γH2AX相关的DNA修复机制来维持基因组完整性。
