The human adenovirus E1A 243R oncoprotein encodes a transcription repression function that localizes to the N-terminal domain (E1A 1-80) and is required for induction of cell cycle progression and neoplastic cell transformation. Our goals are to understand the mechanism of E1A repression in molecular detail and to identify the natural cellular promoters targeted by the E1A repression domain during adenovirus infection. The first specific aim is to define mechanism(s) of E1A repression through in vitro studies using protein-protein interaction and a transcription-repression system. A large panel of EIA single amino acid substitution mutants will be used to define interactions among E1A, p300/CBP, and TBP and to establish their relevance to the E1A repression function. The 3D structure of the E1A N-terminal repression domain will be determined by NMR spectroscopy to help understand the interactions between E1A and its cellular partners. Our working model is that E1A accesses specific cellular promoters involved in growth regulation through p300/CBP as a molecular scaffold,"""""""" where it then can disrupt interaction between TBP and the TATA box. To test this model, preinitiation complexes assembled in vitro and loaded with known amounts of p300/CBP will be analyzed for E1A repressibility. The second specific aim is to define the mechanism of E1A repression in vivo. Transient expression will be used (i) to establish whether E1A can utilize promoter-bound p300/CBP to access specific genes, (ii) to define the molecular determinants of E1A-repressible promoters by chromatin immunoprecipitation (CHIP), and (iii) to analyze the molecular basis of resistance to E1A repression by non-repressible promoters. To provide genetic proof for the role of TBP as an ultimate target of E1A repression, detailed mutational analysis of TBP single amino acid substitution mutants will be performed in vivo and in vitro. Collectively, the findings from in vitro and in vivo studies will allow the development of a detailed molecular model(s) for the mechanism of E1A repression. The third specific aim will identify by CHIP analysis the natural cellular promoters targeted by the E1A repression domain during infection of quiescent human cells. The functional consequences of interaction between E1A and specific cellular promoters will be established by kinetic studies of the gene specific mRNA and protein products.
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