Kumar Ramesh, Cheok Chit Fang
IFOM-p53Lab Joint Research Laboratory, 8A Biomedical Grove, #06-38, Immunos, A*STAR, S138648, Singapore, Singapore.
IFOM, The FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139, Milan, Italy.
Sci Rep. 2017 Dec 12;7(1):17367. doi: 10.1038/s41598-017-16934-w.
RIF1 plays a key role in inhibiting DNA end resection and promoting NHEJ mediated DNA double stand break repair in G1. However, whether SUMOlyation may regulate RIF1 functions is still largely unknown. Here, we report that RIF1 is SUMOlyated in response to DNA damage. We identified PIAS4 as the primary SUMO E3 ligase required for the SUMOylation of RIF1 protein. Mammalian cells compromised of PIAS4 expression, show impaired RIF1 SUMOylation and defective for the disassembly of DNA damage responsive RIF1 foci. Mechanistically, we show that PIAS4 knockdown abrogates UHRF1-dependent ubiquitination of RIF1, compromising RIF1 protein turnover. We detected intense RPA foci that colocalize with RIF1 foci in PIAS4 knockdown cells. These data highlight an important role of PIAS4-dependent regulation of RIF1, likely mediated by SUMOylation, in the disassembly of RIF1 DNA damage response (DDR) foci. We propose that unresolved RIF1 protein at sites of DNA damage in PIAS4-depleted cells largely accumulates in S phase, and subsequently leads to DNA double strand breaks. Therefore, PIAS4 promotes genomic stability by regulating the timely removal of RIF1 from sites of DNA damage.
RIF1在抑制DNA末端切除以及促进G1期非同源末端连接（NHEJ）介导的DNA双链断裂修复过程中发挥关键作用。然而，SUMO化修饰是否调控RIF1的功能在很大程度上仍不清楚。在此，我们报道RIF1在DNA损伤应答时发生SUMO化修饰。我们鉴定出PIAS4是RIF1蛋白SUMO化修饰所需的主要SUMO E3连接酶。PIAS4表达受损的哺乳动物细胞表现出RIF1 SUMO化修饰受损以及DNA损伤应答性RIF1病灶的解聚存在缺陷。从机制上讲，我们发现敲低PIAS4可消除RIF1的UHRF1依赖性泛素化，损害RIF1蛋白的周转。我们在敲低PIAS4的细胞中检测到与RIF1病灶共定位的强烈RPA病灶。这些数据突出了PIAS4依赖性调控RIF1的重要作用，这可能是由SUMO化修饰介导的，在RIF1 DNA损伤应答（DDR）病灶的解聚过程中。我们提出，在PIAS4缺失的细胞中，DNA损伤位点处未解决的RIF1蛋白在很大程度上在S期积累，随后导致DNA双链断裂。因此，PIAS4通过调控从DNA损伤位点及时去除RIF1来促进基因组稳定性。
