Transcriptional modulation by the adenovirus E1A243R oncoprotein
Description
The adenovirus E1A 243R oncoprotein induces DNA synthesis in quiescent cells. E1A-mediated transcriptional activation of cellular genes encoding essential DNA replication proteins is pivotal to the process of activating DNA synthesis. E1A modulates transcription directed by the promoter of the human proliferating cell nuclear antigen (PCNA) gene in a differential manner that is specified by distinct classes of cellular transcriptional activators. Specifically, E1A 243R stimulates or represses PCNA transcription activated by the glutamine-rich activator, Sp1Q, or the acidic activator, RegII, respectively. In the work described here, we explore mutants of E1A to characterize the mechanisms that lead to these diverse responses. Surprisingly, our findings indicate that an intact N-terminus of E1A is required for both activation and repression. These observations suggest that the N-terminus of E1A induces dramatically different effector-dependent transcriptional responses Substituting the PCNA basal promoter with an artificial basal promoter containing the adenovirus E1B gene TATA motif and upstream Gal4 binding sites (G5BCAT) alters the response to E1A 243R. We show here that the N-terminus of E1A mediates the differential transcriptional responses of G5BCAT. The basal promoter sequence from human immunodeficiency virus type 1 (HIV-1) represents a third class of basal promoter elements that responds to E1A modulation. To identify specific basal promoter elements that mediate differential responses to E1A, we prepared chimeras with motifs (TATA and initiator) from the HIV promoter introduced into the background of the PCNA basal promoter (-35 to +15). Although reduced transactivation was observed upon introduction of the HIV initiator motif into the PCNA basal promoter, a more dramatic decrease (from 8-fold to 2-fold) in transactivation was observed upon introduction of the HIV TATA motif. These observations suggest that basal promoter elements can function as selective determinants in response to transcriptional modulation by E1A 243R To identify DNA-protein complexes that mediate these transcriptional responses to E1A 243R, we employed electrophoretic mobility shift assays with the PCNA basal promoter and HeLa cell nuclear extracts. Two major complexes formed with PCNA basal promoter sequences (-35 to +15 relative to transcription initiation at +1), one that is located upstream (C2), and another that is situated around the initiation sequence (C1). Kinetic and competition experiments indicated that bacterially-expressed E1A 243R enhances the rate of complex formation and the stability of both complexes. These results suggest a molecular mechanism by which E1A transactivates the growth-responsive PCNA gene by stabilizing DNA-protein interactions on the TATA-less PCNA basal promoter. (Abstract shortened by UMI.)