去泛素化酶USP54通过参与DNA损伤修复维持基因组稳定性的作用机制

It is known that the timely initiation and termination of DNA damage repair are related to genomic stability and cell fate. Although it has been reported that histone ubiquitination plays an important role in DNA damage repair through contributing to DNA damage response, the molecular mechanism underlying how DNA damage repair is terminated accurately remains to be investigated. By high-throughput screening including interacting proteome and immunofluorescence coupled high-content screening, we found that the deubiquitinated enzyme USP54 acts as a potential histone deubiquitinase to catalyze both H2AK15 and H2BK120 deubiquitination. Interestingly, we found that USP54 is physically associated with the CtBP/LSD1/CoREST complex, and we speculate that USP54 and the CtBP/LSD1/CoREST complex are functionally involved in DNA damage repair process. In the project, we will utilize molecular and cell biology techniques including stable isotope labeling of amino acid (SILAC), laser microirradiation, and micrococcal nuclease sensitivity experiments to further verify the results of high-throughput screening. In addition, we will further investigate the molecular mechanism underlying how USP54 is involved in the accurate termination of DNA damage repair and maintenance of genomic stability, and then explore the biological effects of USP54 in breast cancer. Our study will identify USP54 as a novel histone deubiquitinase, and reveal the molecular mechanism of the crosstalk among histone ubiquitination, methylation, and acetylation in DNA damage response, providing the molecular basis and theoretical basis of USP54 as a potential target for cancer treatment from the perspective of DNA damage repair and epigenetic regulation.

DNA损伤修复的及时触发和按时终止关乎基因组稳定性和细胞命运。组蛋白泛素化等修饰导致的染色质重塑为DNA损伤修复的启动提供场所，而精确终止修复过程的作用机制却知之甚少。我们前期工作通过互作蛋白组和免疫荧光-高内涵等高通量方法，初步发现USP54具有潜在的组蛋白去泛素化酶活性，并能够与CtBP/LSD1/CoREST复合体相互作用，推测他们在DNA损伤修复中协同发挥作用。本项目将利用SILAC、激光微束照射和微球菌核酸酶敏感性实验等分子及细胞生物学技术方法，验证前期筛选结果，进一步探讨USP54参与DNA损伤修复及时终止以维持基因组稳定性的分子机制，探究其在乳腺癌中的生物学效应。本研究将鉴定出USP54是一个新的组蛋白泛素化负调控因子，揭示DNA损伤修复中组蛋白泛素化与甲基化和乙酰化修饰交互对话的分子机制，从DNA损伤修复和表观遗传调控的角度，为USP54作为肿瘤治疗的潜在靶标提供理论依据。

受资助人聚焦表观遗传调控方向，利用分子生物学、细胞生物学及全基因组高通量测序等技术，揭示多种疾病发生发展的表观遗传调控机制。受本项目资助期间，以共同第一或共同通讯作者身份在Cell Metabolism 2022、EMBO Reports 2021和Frontiers in Cell and Developmental Biology 2021发表文章3篇（均标注本项目编号）。研究成果鉴定发现表观遗传修饰酶（组蛋白乙酰转移酶ING5）和转录因子（缺氧诱导因子HIF-1α）的上游功能调控因子，阐释影响上皮向间质转换和脂肪酸氧化等细胞生物学过程的分子机制；此外，围绕物质代谢和表观遗传调控，研究揭示葡萄糖代谢异常导致的糖酵解—组蛋白乳酸化（H4K12la）—PKM2正反馈环路影响细胞稳态的表观遗传机制，为肿瘤等疾病的治疗及药物开发提供分子基础和新思路。