端粒保护蛋白TRF2结合ITS启动衰老的机制- - 调控去磷酸酶PPP2R2C转录表达并激活DNA损伤体系

Cellular senescence induces tissue degeneration and aging syndrome. Telomere dysfunction activates DNA Damage Response (DDR) and leads to senescence. Our previous paper has shown that Telomeric Repetitive Factor 2 (TRF2) as a core protein of multi-enzyme complexes at telomeres can protect telomere during replication by inhibiting DDR machinery. This revealed the mechanism of TRF2 in preventing aging. In this project, we will explore the mechanism of TRF2 and Interstitial Telomeric Sequence (ITS) in the chromatin reprogramming of DDR activation. Our high-throughput Chip-Sequence analysis has shown that TRF2 binds to the ITS at the promoter region of phosphorylase PPP2R2C. We will use (1) Chip assay to verify our proposal that cellular senescence will induce the dissociation of TRF2 from telomeres and recruit it to the ITS; (2) reporter gene system and protein system to show that TRF2 binds to the ITS and induces the trans-regulation of PPP2R2C transcription; (3) genotoxic reagents induced senescence model to show PPP2R2C can phosphorylate H2AX, a crucial sensor protein of DDR system. Our primary experiments have shown that gene silencing of PPP2R2C, followed by RT-PCR and IF assay indicate that TRF2 may control DDR activation and initiate senescence by regulating PPP2R2C expression. Different from the concept of replicative senescence, the novel proposal of DDR induced senescence are due to the cellular micro-environmental changes, which induce the equilibrium of TRF2 expression between telomeres and ITS. TRF2 binding at ITS will participate in the chromatin reorganization and activation of DDR, and finally mediate senescence. This project will contribute to the screening of senescence markers, prevention of organ fibrosis and the treatment of degenerative diseases.

老年性疾病和器官功能退化与细胞衰老有关。传统观念认为端粒复制损伤促进细胞衰老。我们发现端粒参与染色质重组激活DNA损伤（DDR）可能是端粒调控衰老的新机制。我们前期研究证明端粒保护蛋白TRF2为中心的多酶体系抑制DDR，确保端粒复制完整和预防衰老；Chip-Seq分析TRF2能结合至去磷酸化酶PPP2R2C启动子区的间质性端粒（ITS）。本课题将⑴使用Chip等方法证明细胞衰老致TRF2从端粒解离并招募至ITS；⑵报告基因体系等证明TRF2结合ITS反式调控PPP2R2C转录；⑶基因毒性衰老模型证明PPP2R2C能修饰DDR重要蛋白H2AX磷酸化；基因静默等证明TRF2调控PPP2R2C表达的作用为操纵DDR，启动衰老。该研究将探索并阐明环境等致TRF2在端粒和ITS的长距离染色质互动，通过染色质重组激活DDR，是除复制损伤以外的端粒调控衰老的又一新机制，为老年性疾病预防和治疗提供新思路。

本项目严格按计划完成研究内容，运用小鼠模型、斑马鱼基因敲除模型、人肿瘤细胞和原代成纤维细胞模型和人睑板腺肿瘤标本，证明端粒重要蛋白TRF2的端粒外重要作用参与细胞衰老和肿瘤发生。我们首先证明了TRF2在组织中的差异性表达，在组织发育和再生、特别是神经组织的发育和再生中起重要作用，并证明其机制可能通过结合至PPP2R2C启动子区的ITS，调控PPP2R2C的转录表达，影响PPP2R2C蛋白表达和其去磷酸化修饰功能，特别是影响H2AX等 DNA损伤信号通路的重要蛋白的磷酸化修饰，进而激活下游细胞周期稽查点，改变细胞动态平衡，启动衰老。我们还进一步证明了TRF2在细胞衰老中的作用与P53突变失活一起参与了人睑板腺肿瘤的恶性增殖和清晰性的作用。进一步的药物筛选发现并证明，TRF2的存在能增强拓扑异构酶抑制剂ICRF-193对端粒的损伤，而抑制TRF2能减弱拓扑异构酶抑制剂ICRF-193介导端粒损伤和细胞增殖。这些研究作为一个体系证明了端粒特异性蛋白TRF2在端粒和端粒外的作用，共同参与细胞衰老和肿瘤的发生、恶性增殖和侵袭，为我们后续筛选以TRF2为靶点的促进组织再生、抑制肿瘤增殖的药物提供理论基础。.本课题执行期间，已发表标注本项目的通讯/并列通讯文章共计12篇，其中SCI 文章7篇（通讯），核心期刊5篇（通讯），非通讯非第一作者文章3篇。培养及参与培养毕业硕士研究生8名，在读博士研究生4名，在站博士后1名。以第一申请人获得国家专利一项。.此外，本课题的主要研究成果正在投稿国际影响力的杂志上（SCI>10）论著1篇，准备投稿1篇，拟投稿于国际顶级杂志。