RNF20/RNF40/WAC蛋白复合体在DNA损伤反应和肿瘤发生中的作用

Recent work from my laboratory and others has shown that the RNF20/RNF40/WAC complex plays an important role in maintaining genomic stability. Dysfunction of this complex will not only induce genomic instability but also cause a wide variety of human cancers. However, the mechanisms by which the RNF20/RNF40/WAC complex regulates DNA damage repair and what are the tumor suppression functions of the complex are poorly understood at the molecular level. To address these questions, we screened the potential downstream DNA-damage-response regulators of RNF20/RNF40/WAC. Interestingly, we found that following DNA damage, the RNF20/RNF40/WAC complex is required for targeting SAMRCAD1, a newly identified chromatin remodeler participating in DNA damage repair, to DNA damage sites. We also performed unbiased protein affinity purification to search for the functional partners of the RNF20/RNF40/WAC complex. Surprisingly, we found p53 as one of the binding-partners. Our preliminary study suggests that p53 is an upstream functional partner of the complex during transcriptional regulation. With these novel findings, we hypothesize that the RNF20/RNF40/WAC complex regulates chromatin remodeling in various biological processes including DNA damage repair and plays a critical role in the p53-dependent gene transcription. We will test this hypothesis by: determining the molecular mechanism by which the RNF20/RNF40/WAC complex-dependent H2B ubiquitination regulates DNA damage repair and gene transcription (Aim1 and 2); examining the functional defects of cancer-associated mutations of RNF20, RNF40 and WAC (Aim 3).

RNF20/RNF40/WAC复合体对于维持基因组稳定性、抑制肿瘤形成有着重要作用。为探索RNF20/RNF40/WAC调控DNA损伤修复，抑制肿瘤形成的分子机制，我们发现染色质解构因子SMARCAD1的招募行为依赖于RNF20/RNF40/WAC，还发现RNF20/RNF40/WAC同转录因子p53相互作用，并调控靶基因的转录。基于这些线索，我们提出如下假说：RNF20/RNF40/WAC通过招募SMARCAD1在DNA损伤修复过程中调控染色质结构； 依赖于RNF20/RNF40/WAC的H2BK120ub1参与对p53靶基因的转录调控。为验证该假说，本项目计划研究依赖于RNF20/RNF40/WAC的H2BK120ub1调控DNA损伤修复和基因转录的分子机制（研究内容1和2），并进一步探讨与肿瘤相关的RNF20，RNF40和WAC基因变异所造成的生理功能缺陷（研究内容3）。

RNF20/40/WAC复合体对于维持基因组稳定性、抑制肿瘤形成有着重要作用。为探索这一复合体如何调控DNA损伤修复，抑制肿瘤形成的分子机制, 我们（1）研究RNF20/40/WAC和H2BK120ub1 在DNA损伤反应中的生理作用；（2）研究RNF20/RNF40/WAC和p53协同作用调节基因转录的分子机制；（3）研究了LSD1与RNF20/RNF40的相互作用通过调控H2BK120ub1和H3K4和H3K9的单甲基化和二甲基化的水平参与DNA损伤反应的生理机制；（4）研究了WAC如何与sc35在基因转录，R-loop形成及DNA损伤中发挥协同作用的分子机理。此外我们还研究了BRCA2的OB结构域和EXO1的PIN结构域与PAR（多聚 ADP-核糖）相结合参与DNA 损伤修复反应的机制以及BACH1与BCLAF1如何共同参与DNA双链断裂损伤同源重组修复的分子机制。.我们明确了RNF20/40和WAC通过ATR-CHK1通路参与DNA损伤修复通路。确定了RNF20/40和WAC同p53协调作用参与靶基因的转录过程。发现RNF20/RNF40 和WAC一起参与R-loop的形成，并在转录与转录后的剪切过程中也存在协同作用。发现了LSD1在DNA损伤通路中居于RNF20/40和WAC的上游。我们还发现PIN 和OB-fold是两类重要的PAR结合结构域，含有这两种结构域的蛋白如EXO1， hSSB1和 BRCA2等通过与PAR的结合介导其快速募集到DNA损伤位点，以及BRCA1调控BACH1 和BCLAF1参与DNA双链断裂损伤的同源重组修复过程。