The adenovirus early region III (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both basal and E1A-induced transcriptional regulation of the E3 promoter, DNAase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I) relative to the start of transcription. The proteins binding to either region II or region III are required for stabilizing binding to the TATA sequence (site I) and for complete basal and E1A-induced transcription. A study of these E3 transcription factors will be important in understanding the mechanism of E1A transcriptional activation. Oligonucleotide affinity chromatography will be used to purify each of these factors. These purified proteins will be used to make monoclonal antibodies and microsequence analysis performed so that the genes encoding these proteins can be cloned and large quantities of these proteins produced in bacterial expression systems. Potential posttranslational modifications of these proteins in response to the E1A proteins will be studied and the specific E3 sequences with which these proteins interact will be identified. Finally, we will study E3 transcription factors in yeast. The E3 promoter has been introduced into yeast and authentic E3 mRNA start sites obtained. DNAase I footprinting has shown that similar sites are protected in both yeast and Hela cells. Factors binding to these sites will be purified from yeast and compared to the factors in Hela cells. E1A has been produced in yeast and the effects of E1A on the binding of yeast transcription factors will be determined. Yeast genetics will be used to further study these transcription factor genes. These studies should be helpful in understanding the role of the E1A protein in potential alterations in the activity of interactions of cellular transcription factors.
