组蛋白甲基转移酶SETD2蛋白稳定性调控的分子机制及其功能探究

SETD2 is now generally known as the single human methyltransferase responsible for H3K36me3. H3K36me3 recruit histone modification readers to carry out specific processes, including transcription elongation, RNA alternative splicing, DNA repair and so on. The presence of inactivating mutations and deletions of SETD2 in a range of tumor types, most notably in kidney cancer, suggesting a role in tumor suppression. It is not clear how to regulate SETD2 protein levels. In our preliminary study, we found that the stability of SETD2 protein is regulated by ubiquitin-proteasome system. We performed a genome-wide functional gene screening and identify a member of the F-box protein family, SAFP1, for SETD2 protein regulation. In this proposal, we are going to further investigate the molecular mechanism of SAFP1 regulating SETD2 degradation. Also, we plan to study the function of SAFP1 on regulation of H3K36me3, RNA alternative splicing and DNA repair. Furthermore, we are going to explore how SAFP1 and SETD2 might regulate kidney cancer development by using cell models and mouse models. These research is expected to further reveal the molecular mechanism of the regulation of SETD2 protein stability, and provide a new theoretical basis for the diagnosis and treatment of kidney cancer.

SETD2是人体细胞中已知的唯一能够催化H3K36me3的甲基转移酶。H3K36me3能够招募相关识别蛋白发挥调控转录延伸、可变剪接和DNA损伤修复等生物学功能。在多种癌症特别是肾癌中都检测到了SETD2的突变和缺失，因此SETD2被认为是一种抑癌基因。目前对于细胞内SETD2蛋白水平是如何被调控的尚不清楚。我们前期研究发现SETD2受到泛素-蛋白酶体途径的调控。随后利用全基因组范围内的功能基因筛选系统，鉴定出SETD2稳定性的调控因子F-box家族蛋白SAFP1。本项目计划在此基础上，首先探究SAFP1调控SETD2降解的分子机制，以及SAFP1对H3K36me3、可变剪切和DNA损伤修复的调控作用。进而利用细胞模型和小鼠模型探究SAFP1和SETD2在肾癌发生发展过程中的调控作用。我们的研究有望进一步揭示SETD2蛋白稳定性调控作用的分子机制，为未来肾癌的诊断、治疗提供新的理论依据。

SETD2是一种主要的组蛋白H3K36三甲基转移酶，已被证明在多种生物学过程中发挥重要作用。SETD2突变已被证明存在于多种癌症中，包括肾透明细胞癌(ccRCC)。. 基于TCGA数据库和组织芯片，我们发现SETD2突变或低表达与ccRCC患者的不良预后相关。通过CRISPR/Cas9筛选系统和免疫沉淀-质谱分析，我们鉴定了调控SETD2稳定性的E3泛素连接酶SIAH2。随后验证了SIAH2以剂量依赖的方式下调SETD2的蛋白稳定性，并进一步证实了SIAH2通过蛋白酶体依赖途径促进SETD2的降解。同时，我们构建了SIAH2突变体，并证明其降解依赖于其酶活性。此外，SIAH2敲除增加了SETD2的蛋白水平。在SETD2稳定敲低的ccRCC细胞系中，SETD2的缺失增强了细胞的迁移能力，而SIAH2的敲低导致SETD2缺失效应的恢复。此外，SIAH2通过降解SETD2能够调控TRIB3基因附近组蛋白H3K36me3水平，并影响其mRNA m6A修饰，进而影响TRIB3 mRNA稳定性并调控TRIB3基因表达。本研究首次揭示了E3泛素连接酶SIAH2与SETD2相互作用并促进SETD2泛素化降解的机制。此外，SIAH2/SETD2/TRIB3轴可能是肾透明细胞癌潜在的治疗靶点。. 同时在本项目资助下，我们在对蛋白质稳态与肾癌等泌尿系统肿瘤的后续研究中，进一步发现RNF126、PRR11等关键调控因子，发表多篇学术论文。