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

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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.