Combined interaction of PHD and Chromo domains directs NuA4 to DNA double-strand breaks

• Main Authors: Li-ChiaoChia, 賈力橋
• Other Authors: Hung-Jiun Liaw
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/17608464163506365114

碩士 === 國立成功大學 === 生命科學系碩博士班 === 101 === DNA double strand breaks (DSBs) are the most dangerous lesions to the integrity of genome. Failure to repair DSBs properly can lead to genomic instability and cancer. The mechanism of DSB repair is highly conserved from yeast to human. In yeast Saccharomyces cerevisiae, the histone modification complex, NuA4 (functional homolog of Tip60 in human) is recruited to DSBs where it acetylates histone H2A and H4, presumably relaxing the chromatin and allowing the access of repairing proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact with methylated K4 and K36 of histone H3 (H3K4me and H3K36me) by their plant homeodomain (PHD) and chromo domain respectively in vitro. Here, we demonstrated that mutations either in the Yng2 PHD domain (yng2-PHD) or the Eaf3 chromo domain (eaf3-W84A/W88A) have no significant effect on cell growth or DNA repair. However, combined mutations both in the PHD and chromo domain (yng2-PHD eaf3-W84A/W88A) show dramatic defect both in cell growth and DNA damage repair. In addition, the chromatin immunoprecipitation (ChIP) experiments reveal that high level of phospho-S129 of histone H2A (H2AS129p), acetyl-K12 of histone H4 (H4K12ac), H3K4me2, H3K4me3, H3K36me2, and Yng2 are enriched at HO DSB site, suggesting histone H2A-S129p, H3K4me2, H3K4me3, and H3K36me2 are specifically modified at the DSB site and NuA4 is efficiently recruited at the DSB site to acetylate histone H4. By contrast, the eaf3-W84A/W88A yng2-PHD double mutant dramatically reduced the enrichment of H4K12ac at the DSB, suggesting the impairment of NuA4 recruitment. H2A-S129p activation is prolonged at DSB, suggesting the double mutant impairs DSB repair. Our results suggest that the DSB can induce the specific H2AS129p, H3K4me2, H3K4me3, and H3K36me2 modifications at DSB, which in turns recruits NuA4 complex by the multiple interactions between these modified histones and subunits of NuA4. These interactions include H2AS129p-Arp4, H3K36me2-chromo domain and H3K4me3-PHD interactions and these multiple interactions strengthen the recruitment of NuA4 at DSBs.