Transcription factors play a vital role in cell survival, growth and differentiation. They regulate transcription of specific set of target genes in response to extra- and intra-cellular signals. While modulating the expression of desired genes the transcription factors maintain a profile of transcriptional activity within a cell. The cellular transcriptional profile is often affected by overexpression of one or more transcription factors that may result in transcriptional-interference or transcriptional-squelching. The physiological significance of this phenomenon and its effect to the cell is not known. The investigator's recent studies have revealed a profound physiological consequence of AP-2 transcriptional self-interference when AP-2 was induced to overexpress. Transcriptional self-interference of AP-2 induced tumorigenicity in the human teratocarcinoma cell line PA-1. Overexpression of AP-2 also induced tumorigenicity in the murine cell line NIH 3T3. Moreover, N-ras oncogene uses the property of AP-2 transcriptional self-interference to induce tumorigenicity in PA-1 cells. In this study the investigator will characterize the transcriptional-interference phenomenon using AP-2 as a model transcription factor and explore the interesting property, transcriptional interplay with other transcription factors and the physiological consequences. Preliminary experiments suggested that transcriptional coactivators played a critical role in AP-2 transcriptional self-interference and tumorigenicity. Protein-protein interaction studies were carried out to identify the coactivators of AP-2 transcriptional activation. They identified three polypeptides with molecular masses 110 kDa. 74 kDa and 19 kDa that specifically interacted with AP-2. Based on a number of criteria they have identified the 110 kDa polypeptide as poly ADP-ribose polymerase, the 74 kDa protein as RAP74 subunit of transcription factor TFIIF and the 19 kDa protein as the positive coactivator PC4. In this proposal, the coactivator role of these candidate proteins in AP-2 transcription will be characterized. The investigator will select the proteins that have coactivator function and test their ability to relieve AP-2 transcriptional self-interference and their potential to reverse tumorigenicity. As an alternative approach, the N-terminus of AP-2 that contains the self interference property will be investigated. Fine deletion mutations will be made in the N-terminus of AP-2 and analyses will be carried out to localize the amino acid sequence that cause self-interference. The region of AP-2 will be investigated for post translational modifications. As an important goal, additional proteins that interact with the self-interference region will be cloned and analyzed for their role in AP-2 transcriptional activation, tumorigenicity and differentiation. The importance of transcriptional coactivators such as TAFs, positive coactivators (PCs), negative coactivators (NCs) and many others in the transcriptional activity of a wide array of activators is increasingly becoming evident. Most of the studies were carried out in vitro and the nature of these coactivators in pathological conditions has not been investigated. Abolition of AP-2 transcriptional self-interference using coactivators might reverse tumorigenicity of PA-1 cells induced by RAS oncogene and that would clearly establish the clinical importance of transcriptional coactivators.
