A novel regulator of p53 function

描述（由申请人提供）：p53肿瘤抑制基因产物是响应遗传毒性应激的细胞周期停滞和凋亡的关键介质。这些反应对于防止具有不稳定基因组的易转化细胞的出现至关重要。p53作为DNA损伤检查点的生物学作用直接归因于其作为序列特异性转录激活因子的生化功能。事实上，几乎所有与癌症相关的p53突变都会废除这一功能。p53的反式激活功能需要辅助蛋白复合物，即共激活因子，如p300/CBP家族。我们已经确定了hADA复合物，酵母ADA复合物的同系物，作为一种新的p53共激活剂。我们假设，含组蛋白乙酰转移酶（HAT）的hADA复合物，无论是单独还是与p300/CBP协同作用，通过乙酰化p53和p53靶启动子中的染色质相关组蛋白来调节p53介导的DNA损伤反应的程度或性质。确定ADA 3是否是募集ADA复合物至p53的组分，如我们的初步结果所示，并使用诱变来定位介导它们相互作用的ADA 3和p53结构域。我们将使用过表达和显性负性方法来研究ADA复合物是否作为p53的共激活因子发挥作用，并确定共激活因子的功能是否与p53靶基因启动子上的组蛋白乙酰化和/或p53乙酰化有关。最后，我们将量化p53介导的细胞周期阻滞和DNA损伤的凋亡反应，以评估ADA复合物与p300/CBP在调节p53介导的DNA损伤反应中的作用，使用过表达以及显性负性策略。一个成功的结果，拟议的研究应确定一个新的机制，调节p53介导的DNA损伤反应，并提供潜在的目标，为未来的发展诊断剂和合理的治疗人类癌症。

DESCRIPTION (provided by applicant): The p53 tumor suppressor gene product is a key mediator of cell cycle arrest and apoptosis in response to genotoxic stress. These responses are essential to prevent the emergence of transformation-prone cells with unstable genomes. The biological role of p53 as a DNA damage checkpoint is directly attributable to its biochemical function as a sequence-specific transcriptional activator. Indeed, nearly all cancer-associated p53 mutations abrogate this function. The transactivation function of p53 requires accessory protein complexes, the coactivators, such as the p300/CBP family. We have identified hADA complex, the homologue of the yeast ADA complex, as a novel coactivator of p53. We hypothesize that histone acetyl transferase (HAT)-containing hADA complex, either by itself or in concert with p300/CBP, regulates either the extent or the nature of p53-mediated DNA damage response by acetylating p53 and chromatin-associated histones in p53 target promoters.To address these hypotheses, we will first characterize the components of human ADA complex in cells under study, determine if ADA3 is the component that recruits ADA complex to p53, as our preliminary results suggest, and use mutagenesis to map the ADA3 and p53 domains, that mediate their interaction. We will use overexpression and dominant-negative approaches to investigate if ADA complex functions as a coactivator for p53, and determine if coactivator function relates to histone acetylation on p53 target gene promoters and/or p53 acetylation. Finally, we will quantify p53-mediated cell cycle arrest and apoptotic responses to DNA damage to assess the role of ADA complex versus p300/CBP in regulating p53-mediated DNA damage response, using overexpression as well as dominant-negative strategies. A successful outcome of the proposed studies should identify a novel mechanism for regulating p53-mediated DNA damage response, and provide potential targets for future development of diagnostic agents and rational therapeutics for human cancer.