The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40 -80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.Group C adenovirus (Ad) 1 E1A encodes two multifunctional regulatory proteins of 243 and 289 amino acid residues (243R and 289R). The E1A proteins are involved in diverse cellular functions, including transcriptional activation, transcriptional repression, induction of cellular DNA synthesis, cell immortalization, cell transformation, as well as inhibition of metastasis and of cell differentiation (for reviews, see Refs. 1-5). E1A 243R differs from E1A 289R only by conserved region 3 (CR3), a 46-amino acid domain unique to 289R. E1A is the first viral gene expressed during productive infection of permissive human cells and is required to activate transcriptionally early viral genes. CR3 is essential (6 -10) and sufficient (11-12) for transactivation of early viral genes.The 243R protein encodes domains required for the growth regulatory functions of E1A. An intriguing function of 243R is its ability to repress transcriptionally a set of cellular genes involved in growth regulation and differentiation (13-17), as well as several viral promoters including those of SV40, polyoma virus, and human immunodeficiency virus type 1 (18 -20). How the E1A repression function interfaces with its growth regulatory properties is not known. Furthermore, the molecular mechanism of transcriptional repression is not understood. Elucidation of mechanism would inform our understanding of the biological roles of E1A transcriptional repression.One can imagine several mechanisms by which E1A might repress the activity of a target gene (for review, see Ref. 21). For example, E1A could (i) bind to promoter DNA and block access by a transcriptional activator; (ii) sequest...