The investigators would like to extend their understanding of the mechanisms by which HMG-1 protein participates in the regulation of gene expression by RNA pol II. HMG-1 represses basal level transcription, while enhancing the level of transcription activated by Oct-1 and USF. They will determine the nature of the HMG-1 interactions with general transcription factors, TBP and TFIIB; activator oncoprotein E1A; and the products of tumor suppressor genes, Rb and p53. The binding will be examined both in the presence and in the absence of a DNA promoter. Of particular interest will be the competitive interactions which the HMG proteins engage in with other transcription factors in the process of exerting their influence to control gene expression and the regulation of the cell cycle. Protein-protein interactions of HMG-1 with regulatory proteins will be examined using GST-fusion capture experiments and analyzed by SDS-PAGE and western blots. A major aim will be to provide an experimental basis which begins to explain the apparent dichotomy that HMG-1 serves as a repressor for basal level transcription, while acting to enhance activated transcription in other characterized promoter systems. HMG-1 binds to the TBP/TATA complex to produce a transcription """"""""dead end"""""""" complex, which we believe may be competed off by activators. In addition, the occurrence of protein-protein interactions in solution may play an important, but indirect role. HMG-1 may interact with an activator in increase its effectiveness so that activators would interact more effectively with GTFs and stimulate the level of transcription. The activator proteins may then serve both as an anti-repressor and activator, while HMG-1 would serve as either a repressor and/or coactivator, depending on the circumstance. The investigators shall use findings with E1A and p53 as a tentative model to compare with. On the other hand, HMG-1 may serve as a cellular activator by competing with the E2F regulator transcription factor to stimulate the dissociation of the repressor, Rb, from the Rb/E2F complex. HMG-1 has the minimal recognition sequence for binding to the """"""""A/B box"""""""" in the Rb protein. They will map the region(s) of HMG-1 and Rb involved in the direct interaction. By its complexation with both TBP and Rb, HMG-1 may play a pivotal """"""""linking role"""""""" in the concerted regulatory action between transcriptional regulation, the control of the cell cycle, cell proliferation and the development of cancer. It is important to note in this respect that both HMG-1 and E2F bind to both TBP and Rb and that the interaction of E2F and Rb are mutually exclusive. The finding from the proposed studies shall provide additional new insights into the possible mechanisms of action for HMG-1 protein in the regulation of gene expression in normal and unregulated cells.
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| Das, D; Scovell, W M (2001) The binding interaction of HMG-1 with the TATA-binding protein/TATA complex. J Biol Chem 276:32597-605 |
