SUMO E3-Ligase Regulates DNA Damage-Dependent Exchange of the Histone Variant H2A.Z-2

Abstract

Recently, in the regulation of DNA damage response, the importance of the exchange of histone variants as one of the important mechanisms for reorganization of damaged chromatin has been recognized. In addition, the small ubiquitin-related modifier (SUMO) system has been shown to play an important role in the posttranslational modification of DNA repair proteins. Recent studies have implicated yeast H2A.Z in the DNA repair process and reported the presence of two H2A.Z isoforms, H2A.Z-1 and H2A.Z-2, in vertebrates. However, the role of each of these vertebrate H2A.Z isoforms in the reorganization of damaged chromatin is still unclear. In this study, we examined the role of human H2A.Z isoforms in the reorganization of chromatin after the induction of DNA double strand breaks (DSBs) and the importance of SUMOylation in its mechanism of action. To examine the dynamics of H2A.Z isoforms at damaged sites, we established GM0637 cells that stably expressed either one of the GFP-H2A.Z isoforms and performed fluorescence recovery after photobleaching (FRAP) and inverse FRAP (iFRAP) analyses in combination with microirradiation. In FRAP analysis, the dynamics of H2A.Z isoform following DNA damage were quantified by determining the fluorescence recovery of the GFP signal within two independent areas – one area in the irradiated region and the other in the unirradiated region of a single nucleus – immediately following microirradiation. In iFRAP analysis, immediately following microirradiation, all fluorescence except for small regions in irradiated and unirradiated areas were bleached and the remaining GFP fluorescence was chased. Finally, to investigate the pathway that regulates H2A.Z isoform dynamics in the damaged chromatin, we performed FRAP and iFRAP analysis with cells expressing siRNA against SUMO E3-ligase, PIAS1 or PIAS4. In FRAP analysis, the fluorescence intensity of GFP-H2A.Z-2 rapidly recovered in the irradiated region, while that in the unirradiated region did not change during the observed time. In contrast, GFP-H2A.Z-1 signal intensity did not show any remarkable changes. In iFRAP analysis, slow diffusion of GFP-H2A.Z-2 into the bleached area was observed within the irradiated area, but not in the unirradiated area. Similar to FRAP analysis, GFP-H2A.Z-1 signal intensity did not show any remarkable changes. These findings indicate that vertebrate H2A.Z-2 is exchanged at damaged sites immediately after the induction of DSBs. The increase in the mobility of H2A.Z-2 after microirradiation was repressed by knockdown of PIAS4, but not PIAS1. We found that vertebrate H2A.Z-2 is involved in the regulation of DNA damage response at a very early stage via reorganization of damaged chromatin. In addition, SUMOylation may affect the regulation of H2A.Z-2 dynamics upon DSBs.