Understanding the Role of TAF1 Double-Bromodomain in p53-mediated Transcription

描述(申请人提供)：P53是一种重要的肿瘤抑制蛋白。它在DNA损伤时被激活，作为参与G1细胞周期停滞和凋亡的基因的转录因子发挥功能。TAF1是转录因子TFIID的最大亚基。TAF1含有两个串联的溴结构域(DBrD)，已知它们在转录调控中与乙酰化赖氨酸相互作用。在这项研究中，我们试图了解TAF1及其DBrD在转录调控中的作用，特别是在P53介导的转录中。这项提议有三个具体目标。在目标1中，将使用Tet诱导的RNAi方法来敲除U2OS细胞中的内源性TAF1，并产生用于AIMS 2和3的DBrD突变细胞系。在目标2中，我们通过进行全基因组微阵列分析和RT-PCR来鉴定DBrD依赖的基因，试图了解TAF1 DBrD在转录调控中的作用。针对p53依赖基因的微阵列研究将在Aim 3下使用紫外线照射细胞的RNA样本进行，以识别需要功能性DBrD转录的p53依赖基因。芯片分析将在AIM 3下进行，以验证观察到的TAF1 DBrD和P53在P53依赖启动子上的相互作用。由于P53在细胞生长和存活中的重要作用，了解TAF1DBrD在P53介导的转录中的作用是相关的。拟议的目标被认为揭示了关于转录对DNA损伤的分子机制的新见解。这些发现为我们深入了解细胞生长控制提供了有价值的信息，从而对不断寻找有希望的人类癌症治疗方法产生了影响。P53是一种重要的肿瘤抑制蛋白。TAF1是一种重要的基因转录蛋白，它通过TAF1双溴结构域与P53相互作用，导致其与p21启动子(细胞生长和存活的重要基因)的相互作用。了解这种相互作用将为控制DNA损伤后细胞生长和存活的分子机制提供新的见解，从而为不断寻找人类癌症的潜在治疗方法做出有希望的贡献。

DESCRIPTION (provided by applicant): p53 is an important tumor suppressor protein. It becomes activated upon DNA damage to function as a transcription factor of genes involved in G1 cell cycle arrest and apoptosis. TAF1 is the largest subunit of transcription factor TFIID. TAF1 contains two tandem bromodomains (double-bromodomain, DBrD), which are known to interact with acetylated lysine in transcription regulation. In this study, we seek to understand the role of TAF1 and its DBrD in the regulation of transcription, especially p53-mediated transcription. There are three specific aims for this proposal. In Aim 1, a Tet-inducible RNAi approach will be used to knockdown endogenous TAF1 in U2OS cells and generate DBrD mutant cell lines to be used in Aims 2 and 3. In Aim 2, we seek to understand the role of TAF1 DBrD in transcription regulation by undertaking genome wide microarray analysis followed by RT-PCR to identify DBrD-dependent genes. Microarray studies specific for p53-dependent genes will be performed under Aim 3 using RNA samples from UV-irradiated cells to identify p53-dependent genes that require functional DBrD for their transcription. ChIP assays will be conducted under Aim 3 to verify the observed interaction between TAF1 DBrD and p53 on p53-dependent promoters. Understanding the role of TAF1 DBrD in p53-mediated transcription is relevant due to the important function of p53 in cell growth and survival. The proposed aims are thought to reveal new insights about the molecular mechanisms governing transcription upon DNA damage. These findings represent valuable information to our in-depth understanding of cell growth control, thus having an impact on the constant search for promising treatments of human cancer. p53 is an important tumor suppressor protein. TAF1, an important protein in gene transcription, has been shown to interact with p53 via TAF1 double-bromodomain resulting in its recruitment to p21 promoters (an important gene in cell growth and survival). Understanding this interaction will provide new insights about the molecular mechanisms that control cell growth and survival upon DNA damage, thus representing a promising contribution to the constant search for potential treatments of human cancer.