锌指蛋白质特异识别（亚）端粒DNA的结构机制

For the last few decades, the only proteins known to specifically bind telomeres were the telomerase enzyme and a protein complex known as the Shelterin complex. Recently, several zinc finger proteins have been identified as new (sub-)telomeric DNA binding proteins which play important roles in telomere length homeostasis. TZAP, a C2H2 zinc finger protein, binds to double-strand telomeric DNA and stimulates telomere trimming. Our recent work which was published on Cell Research provides the molecular basis for the recognition of telomeric DNA by TZAP. Very recently, our collaborator have identified another C2H2 zinc finger protein ZBTB10 which also directly binds to double-strand telomeric DNA, however through a distinct mechanism compared to TZAP as revealed by our preliminary result. In this project, we are planning to investigate the mechanism by which ZBTB10 specifically interacts with telomeric DNA. In another work, we present the crystal structure of the NuRD (The nucleosome remodeling and histone deacetylase) subunit RbAp48 bound to C2H2 zinc finger protein ZNF827 which is important for NuRD activity at ALT telomeres. However, it is still unknown that how ZNF827-NuRD axis is recruited to ALT telomeres. ZNF827 and the two orphan nuclear receptor TR4 and COUP-TF2 may play essential roles in the pathway by directly binding to ALT telomeric DNA. In this project. We are planning to investigate the molecular mechanisms by which the C2H2 zinc fingers of ZNF827 and C4-type zinc fingers of TR4 and COUP-T2 bind to ALT telomeres.

近期发现了若干锌指蛋白质可以直接结合（亚）端粒DNA并调节端粒长度内稳态。TZAP是C2H2类型锌指蛋白质，其直接结合端粒DNA并促进端粒修剪（Telomere Trimming）。我们近期的工作阐述了TZAP特异识别端粒DNA的结构基础。我们在前期研究中发现了另一个亚端粒DNA结合锌指蛋白ZBTB10，我们拟对ZBTB10展开结构生物学研究，阐述其新颖的亚端粒DNA识别机制。在另一项工作中，我们揭示了锌指蛋白ZNF827与染色质重塑复合物NuRD相互作用的结构基础及其在端粒延伸替代机制（ALT）所起的作用。但是ZNF827-NuRD如何定位到ALT端粒区域仍缺少研究。TR4和COUP-TF2核受体锌指蛋白质以及ZNF827可能参与了这一途径的（亚）端粒识别。在本项目中，我们将对TR4和COUP-T2的C4锌指以及ZNF827的C2H2锌指结合（亚）端粒的机制展开研究。

端粒DNA结合蛋白质直接结合于双链端粒DNA区域，并募集下游蛋白质对端粒稳态调节产生影响。近年来的研究发现C2H2锌指蛋白质是新型的端粒DNA结合蛋白质，并参与端粒长度稳态调节。TZAP是一个BTB-ZF家族的C2H2锌指蛋白质，其直接结合端粒DNA并促进端粒修剪过程（telomere trimming）（Science 2017）。申请人解析了TZAP特异识别端粒DNA的结构基础，明确了TZAP特异识别端粒DNA的分子机制（Cell Res. 2018，最后通讯）。本项目立足于C2H2锌指蛋白质特异结合端粒DNA的结构机制，并与其他课题组展开合作，阐述锌指蛋白质结合端粒DNA调节端粒稳态的作用机制。.我们与合作者（Dennis Kappei课题组）鉴定了一枚亚端粒DNA TTGGGG序列的结合蛋白质ZBTB10（Nucleic Acids Res. 2019, 参与作者）。ZBTB10通过串联的两个C2H2锌指(ZF1-2)与双链亚端粒重复序列TTGGGG特异结合。在本项目中，我们解析了ZBTB10 ZF1-2与双链DNA序列TTGGGG的复合物晶体结构。结合等温量热滴定实验，我们确定了ZBTB10 ZF1-2中识别TTGGGG的关键氨基酸残基，并明确了ZBTB10 ZF1-2特异性识别TTGGGG的分子机制。基于该结构，我们进一步理性改造ZBTB10，使其特异识别端粒TTAGGG DNA序列，并解析了高分辨率晶体结构。基于这些结构我们阐述了C2H2锌指识别端粒DNA的一般特征，并发现基因组编码多个C2H2锌指蛋白质具备潜在的端粒DNA结合能力，为后续新型端粒DNA结合蛋白质的发现鉴定和功能研究奠定了基础（JBC 2023, in press，最后通讯）。.我们进一步与Dennis Kappei课题组合作鉴定发现ZNF524的ZF1-4特异识别TTAGGG端粒DNA序列。我们解析了ZNF524 ZF1-4与端粒DNA的高分辨率复合物结构，阐述其特异识别的结构基础。我们进一步发现ZNF524在体内特异结合于端粒区域，并保护端粒免受DNA损伤。该工作发现了一个新型的端粒DNA结合蛋白质，并初步阐述其生理意义，为后续的研究奠定了基础（BioRxiv, 2022, Nat. Commun., in revision，共同通讯）。