基因转录是由一系列转录因子和转录辅助因子参与的复杂过程。转录调控异常会导致多种疾病包括肿瘤的发生与发展。KRAB型锌指蛋白是哺乳动物最大的转录调控因子家族，虽然对其转录抑制的机制有所认知，但其多数成员的靶基因及生物学活性仍未知，主要原因之一是寻找锌指蛋白的DNA结合位点受限。我们在前期工作中克隆到一编码KRAB型锌指蛋白的基因ZNF75D。初步功能分析表明ZNF75D定位于细胞核，具有转录抑制活性且与转录中介因子TIF1β/KAP1及去乙酰化酶存在相互作用。本课题拟从利用ChIP-Seq技术高通量寻找ZNF75D全基因组结合位点和利用蛋白亲和层析联合质谱分析寻找ZNF75D相互作用蛋白入手，深入探讨ZNF75D参与转录调控的机理和生物学功能及其与肿瘤发生发展的关系，为进一步认识KRAB型锌指蛋白在基因转录调控中的作用及其与肿瘤发生发展的关系提供理论依据，为寻找新的肿瘤治疗靶点提供线索。

Krüppel-associated box （KRAB）-containing zinc-finger proteins（KRAB-ZFPs） contains the largest family of transcriptional regulators and over 400 KRAB-ZFPs are encoded in mammalian genomes.While the molecular mechanism of transcription regulation by KRAB-ZFPs has been clarified to some degree，little is known about their biological function or gene targets，which could be largely attributed to the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In this proposal, our work focused on the identification and functional analysis of a KRAB-containing zinc-finger protein, ZNF75D, which contains SCAN, KRAB A and 5 C2H2 zinc finger domains. We demonstrated that ZNF75D is localized in nucleus and possesses intrinsic transcription repression activity. We also found that ZNF75D interacts with TIF1β (transcriptional intermediary factor 1β, also named KAP1 (KRAB-associated protein 1)), histone deacetylase HDAC1 and HDAC2, indicating ZNF75D maybe act as a transcription repressor by recruiting TIF1β/KAP1 and histone deacetylase complexes. Based on these interesting findings, to further elucidate its function, we endeavour to indentify putative DNA-binding sequences and potential downstream targets of ZNF75D by CASTing (Cyclic Amplification and Selection of Target) assays and ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing). To further understand the molecular mechanisms underlying ZNF75D-mediated transcription repression, the ZNF75D-containing protein complexes will be identified by affinity purification and mass spectrometry. With the target genes of ZNF75D, its possible roles in tumorigenesis and tumor progression will be further studied. These findings may shed new light on transcriptional regulation by KRAB-ZFPs and might offer a potential new target for tumor therapy.

乳腺癌是女性发病率较高的恶性肿瘤之一，发病率和死亡率居妇女各类恶性肿瘤之首，然而乳腺癌发生发展的分子机制目前尚不完全清楚。锌指蛋白是哺乳动物最大的转录调控因子家族，近年研究发现锌指蛋白在肿瘤形成、生长以及转移中起着重要作用。但对于人类基因组中存在的如此大量的锌指蛋白，它们每种具体的生物学功能仍知之甚少。高通量分析显示编码KRAB型锌指蛋白的ZNF75D和具有PR/SET结构域的锌指蛋白ZNF298（PRDM15）在乳腺癌组织中高表达，但其在乳腺癌中的生物学作用及机制仍未见报道。本课题以乳腺癌为模型，对ZNF75D和PRDM15进行了深入的分子机制及生物学功能探讨。我们研究发现ZNF75D定位于细胞核，具有转录抑制活性且与转录中介因子TIF1β/KAP1、去乙酰化酶和PRDM15等存在相互作用。进一步我们证实ZNF75D和PRDM15均通过促进G1-S周期的进程及抑制细胞凋亡而促进乳腺癌生长增殖。对Oncomine数据库及我们收集的乳腺癌标本进行分析发现ZNF75D和PRDM15在乳腺癌中高表达。通过分析生存曲线，我们发现PRDM15的高表达与乳腺癌较差的预后有关。体外实验发现PRDM15是一个细胞周期相关蛋白，通过促进乳腺癌细胞从G0/G1期进入S期，从而促进乳腺癌细胞增殖。Annexin V/PI染色结合流式细胞检测显示PRDM15沉默加剧VP-16诱导的MCF-7细胞的凋亡。沉默PRDM15后，p53蛋白水平会显著升高，其下游靶基因PUMA、p21等也随之升高，细胞凋亡比例明显增加。但我们研究发现PRDM15虽然具有转录抑制活性，但其过表达及沉默并不影响p53 mRNA水平。用放线菌酮捕获实验发现，当敲低PRDM15以后能够导致p53的半衰期明显延长。以上结果提示我们PRDM15负调控p53并不是影响p53转录水平而是通过间接或直接作用影响了p53的稳定性。总之，我们发现锌指蛋白PRDM15和ZNF75D在乳腺癌发生发展中起重要作用，这一成果不仅丰富了我们对锌指蛋白的认识，也为乳腺癌发生发展机制提供了新的视角，为乳腺癌诊断、治疗和预后提供新的线索。