RNF126在DNA双链断裂修复中的作用

RNF126 is an E3 ubiquitin ligase. The functions of RNF126 in the repair of DNA double strand breaks (DSBs) have not been reported. Our preliminary data suggest that RNF126 plays an important role in both homologous recombination (HR) and Alt-NHEJ (alternative non-homologous end joining) via E3 ligase independent mechanisms. In this study, we will identify the mechanisms by which RNF126 promotes DNA DSBs repair and test if RNF126 promotes HR and Alt-NHEJ via transcription dependent and independent pathways in which E2F1 is involved. We hypothesize that RNF126 promotes the expression of BRCA1 and HR via transcriptionally regulating E2F1. In addition, RNF126 facilitates Alt-NHEJ via promoting ATM dependent RNF126 phosphorylation via direct binding to E2F1. We are going to test this hypothesis via determining if RNF126 binds to the E2F1 promoter via ChIP-qPCR and gel shift assays. A cell free phosphorylation assay and GST pull-down assay will also be utilized to elucidate if RNF126 is phosphorylated by ATM. Last, we will determine the efficacy of Chk1 inhibitor in treatment of cancer cells with RNF126 deficiency using xenograft models. Our expect results will not only provide the evidence suggesting that RNF126 promotes DSBs response but also provide a potential therapeutic target for cancer therapy. Therefore, our proposal study will have a significant impact on both cancer biology and cancer therapy.

E3泛素连接酶RNF126与DNA双链断裂损伤(DSBs)修复的关系未见报导。预实验证实RNF126在同源重组(HR)及替代性非同源末端连接(Alt-NHEJ)修复中具有作用。本项目探讨RNF126促进DSB修复的机制，检验RNF126是否通过转录依赖和非转录依赖机制分别调控HR与Alt-NHEJ修复，但都需要E2F1介导，即RNF126依赖E2F1转录调控BRCA1，促进HR；而通过磷酸化作用与E2F1结合，促进Alt-NHEJ。以ChIP-qPCR及无细胞系统凝胶迁移实验，检测其与E2F1启动子是否直接结合；无细胞磷酸化实验证实ATM磷酸化位点T191，该位点持续磷酸化pull-down实验检验其是否与E2F1直接作用。此外用异种移植裸鼠验证联合方案治疗RNF126缺失肿瘤的必要性。该研究不仅提出RNF126是抑癌基因，又是肿瘤治疗的潜在靶点，为RNF126缺失相关肿瘤的治疗提供依据。

本项目进一步探究了RNF126的生物学功能，并为乳腺癌治疗提供了一个新的靶点。首次提出并证实了 RNF126通过调节BRCA1的表达来促进HR，RNF126作为E2F1的调控因子，通过与E2F1直接作用，促进BRCA1启动子上E2F1的反式激活效应。并且，RNF126的E3连接酶活性对RNF126介导的BRCA1/HR调节并不重要。在我们研究RNF126在ALT-NHEJ修复过程中的作用时，发现RNF126并非依赖ATM磷酸化调控ALT-NHEJ修复过程。这与我们最初的设想不同，进一步研究发现RNF126依赖MRN复合体促进ALT-NHEJ修复过程。同时我们发现R6的表达与细胞的IR暴露成正比，IR可增强细胞R6蛋白的稳定性，R6蛋白的130-140aa区域对于维持R6蛋白的稳定性至关重要。这为我们提供新的研究角度。.浸润性乳腺癌组织中高表达RNF126蛋白。RNF126蛋白高表达是浸润性乳腺癌患者预后不良的独立预后因子，RNF126高表达的乳腺癌患者预后差。RNF126可作为癌肿细胞对CHEK1抑制反应敏感性的潜在生物标志物。通过增强癌细胞内的复制压力，CHEK1抑制剂可特异性靶向RNF126高表达的乳腺癌细胞，提高此类患者生存率。.