CELLULAR PROTEINS INVOLVED IN ADENOVIRUS E1A REPRESSION

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The adenovirus E1A oncogenes encodes two porly understood biochemical activities that map in ElA protein domains important for cell transformation - transcriptional repression and cellular DNA-synthesis induction. It is likely that interaction of cellular regulatory protein(s) with specific EIA protein sequences play critical roles in these EIA oncogene functions. To further understand EIA repression and to develop assays to identify and purify cellular protein factors, we will investigate as models two contrasting and unique enhancer- dependent, ElA repressible genes - the rat insulin II gene and the rat neu oncogene. Cellular factor(s) required for ElA repression of insulin are cell-type specific whereas those for neu are not. Conserved EIA protein domain 2 is reported to be required for neu oncogene repression but to be dispensible for insulin repression. We propose to determine whether cellular protein synthesis is required for EIA repression of insulin and neu by a cell microinjection assay and to study ElA domain requirements for ElA repression of insulin and neu represssion, as well as for EIA DNA-synthesis induction. Comparison of results with those of similiar studies with the ElA repressible SV40 enhancer will (i) help formulate models of EIA repression, (ii) help determine whether two different mechanisms are involved in the EIA repression phenomena - one representing "direct transcriptional repression" and the other possibly reflecting a secondary event in an EIA induced pathway of cellular DNA synthesis, and (iii) guide studies on mechanism and the development of strategies for isolation of cellular protein factors. Our major effort will then be (i) to develop in vitro and in vivo complementation assays for cellular proteins involved in EIA repression, (ii) to identify by several approaches, particularly by EIA peptide-affinity chromatography, cellular proteins that associate with ElA domains and/or function in EIA repression, and (iii) to purify cellular factors that function in EIA repression and to clone their genes for detailed studies.