Convergent functions of p53 and BRCA1 in gene regulation

肿瘤抑制蛋白p53是一种转录因子，参与细胞对DNA损伤的反应，介导生长停滞或细胞凋亡。肿瘤抑制因子BRCA1的特征不太明确，但研究表明，BRCA1的过表达也会导致生长停滞或细胞凋亡，p53和BRCA1已被证明在生理和功能上相互作用。申请中提出的研究旨在探索p53和brca1依赖性转录激活的细节，阐明这些肿瘤抑制蛋白的相互作用是否具有生物学相关性。初步研究通过检测单克隆抗体moAb421在电泳迁移转移试验中对p53序列特异性DNA结合的影响，确定了两类p53应答元件。用moAb421孵育可增强p53与一组应答元件的结合，同时抑制其与另一组应答元件的结合，这表明不同构象的p53可能与每个应答元件子集相互作用。BRCA1选择性地增强了一个位点子集的p53依赖性激活。我们的研究还表明，ATF1转录因子可以在物理和功能上与BRCA1蛋白相互作用，BRCA1可以通过环AMP反应元件（CRE）增强转录。已经确定了包含CRE和p53结合位点的启动子。因此，我们提出的实验目标包括：(1)确定通过单克隆抗体moAb421的差异效应分类的p53应答元件的不同亚群的意义。(2)确定BRCA1如何通过CRE改变转录，并表征BRCA1与ATF1和CREB转录因子成员的相互作用。(3)表征BRCA1和p53在同时包含CRE和p53位点的基因上的相互作用。(4)确定BRCA1和ATF1在p53介导的DNA损伤应答中的作用。由于高达30%的乳腺癌病例中p53发生突变，而BRCA1基因约占遗传性乳腺癌的50%，因此确定这两种肿瘤抑制蛋白是否在参与细胞对DNA损伤的反应以及乳腺癌发病机制的基因表达中发挥趋同作用将是很重要的。

The tumor suppressor protein p53 is a transcription factor that has been implicated in the cellular response to DNA damage and mediates either growth arrest or apoptosis. The tumor suppressor BRCA1 is less well- characterized but over-expression of BRCA1 was shown to also alternatively lead to growth arrest or apoptosis p53 and BRCA1 have been shown to interact both physically and functionally. The research proposed in the application is designed to explore the details of p53 and BRCA1-dependent transcriptional activation and elucidate whether the interaction of these tumor suppressor proteins has biological relevance. Preliminary studies have identified two classes of p53 response elements by examining the effect of monoclonal antibody moAb421 on the sequence-specific DNA binding of p53 in electrophoretic mobility shift assays. Incubation with moAb421 enhances the binding of p53 to one set of response elements by inhibits binding to another set, suggesting that different conformations of p53 may interact with each subset of response elements. BRCA1 selectively enhances p53-dependent activation of one subset of sites. Our studies have also shown that the ATF1 transcription factor can interact with BRCA1 protein both physically and functionally and that BRCA1 can augment transcription through a cyclic AMP response element (CRE). Promoters have been identified which contain both a CRE and a p53 binding site. Thus, the proposed aims include experiments to: (1) Determine the significance of distinct subsets of p53 response elements classified by the differential effect of monoclonal antibody moAb421. (2) Determine how BRCA1 alters transcription through the CRE and characterize the interaction of BRCA1 with members of the ATF1 and CREB transcription factors. (3) Characterize the interaction of BRCA1 and p53 on genes that contain both a CRE and a p53 site. (4) Determine the role of BRCA1 and ATF1 in the p53- mediated response to DNA damage. As p53 is mutated in up to 30% of cases of breast cancer and the BRCA1 gene is responsible for approximately 50% of inherited breast cancer, it will be important to determine whether these two tumor suppressor proteins play convergent roles in gene expression involved in the cellular response to DNA damage as well as in the pathogenesis of breast cancer.