基于内源基因标签模型的BRCA1抑癌机制探究

BRCA1 (breast cancer susceptibility gene 1) is one of the most important tumor suppressors that implicated in hereditary and familial breast cancer, yet little is known about how it function as tumor suppressor in a tissue-specific manner. To explore BRCA1 tumor suppressing function，we endogenously tagged Brca1 C-terminal with Halotag-flag dual tags in mouse haploid embryonic stem cells (haESCs) followed by intracytoplasmic haESCs injection (ICAHCI), generating Brca1 gene tagged mouse model. By tandem affinity purification (TAP), chromatin immunoprecipitation sequencing (CHIP-Seq), and RNP immunoprecipitation sequencing (RIP-seq) in stem cells and in animal tissues. We try to dissect the mechanisms of how Brca1, as genome caretaker, involved in DNA damage repair, cell survival and proliferation. We have initially identified MPG as a BRCA1 E3 substrate in nucleus by TAP, which may play roles in mediating or regulating BRCA1’s physiological function. Furthermore we will perform mechanistic study by combining breast cancer mouse model and clinic samples, hoping to identify novel therapeutic targets and provide new intervention strategy for diagnosis or treatment of breast cancer.

乳腺癌易感基因BRCA1是目前发现最重要的家族遗传性乳腺癌抑癌基因之一，然而人们对 其组织特异性抑癌机制知之甚少。为了加深对BRCA1抑癌功能的理解，我们利用Crispr/Cas9基因编辑技术在小鼠单倍体胚胎干细胞中对Brca1基因C末端进行Halotag-flag标记，通过细胞注射胚胎移植获得基因标记小鼠，在干细胞和动物组织水平纯化蛋白复合物、染色质沉淀高通量测序及结合RNA高通量测序来鉴定新的作用蛋白、染色质调控元件和可能结合的RNA转录产物，发掘Brca1作为“监护者”在DNA损伤修复、细胞生存繁殖等生理功能中的作用。通过TAP纯化我们鉴定BRCA1在核内的E3酶底物MPG，并开始研究其介导或调控的BRCA1具体生理功能。我们将进一步结合小鼠乳腺癌模型和临床样本进行探究，解析BRCA1组织特异性抑癌机制，为乳腺癌等肿瘤的临床诊治开发新的药物靶点和干预策略。

乳腺癌易感基因BRCA1的组织特异性抑癌机制研究意义重大，我们通过Halotag-flag内源性标记的BRCA1蛋白复合物纯化从小鼠胚胎干细胞和乳腺组织中鉴定出MPG和WSB1两个BRCA1相互作用蛋白，并进行了较为深入的相互调控机制研究。BRCA1可能通过泛素化调控MPG在BER通路中的功能，但我们没有鉴定出影响其功能的具体泛素化位点；原癌基因WSB1能在多个细胞系中显著影响BRCA1的蛋白表达和稳定性，生化结果表明WSB1通过泛素化蛋白酶体途径降解BRCA1，并且调节其在细胞周期中的表达，跟WSB1的原癌症功能及BRCA1的抑癌功能相吻合；我们建立了K14-Cre:Brca1f/f乳腺癌模型，将进一步在这个模型中研究MPG及WSB1和BRCA1的相互作用对乳腺癌发生发展的影响。