The transcription factor, EIIF, controls some RNA polymerase II -transcribed genes that are induced in growing cells, such as c-myc, the EGF receptor, and dihydrofolate reductase. It was first identified as a host cell-encoded factor that binds to the Adenovirus E2A promoter. EIIF can be found in a complex that includes the retinoblastoma gene product (Rb) and cyclin A. Rb and cyclin A binding appear to inhibit EIIF-dependent transcription. Rb and cyclin A are released from EIIF in response to cell cycle control signals, or upon binding of Rb or cyclin A by tumor virus oncogenes such as SV40 large T antigen or Adenovirus EIA. We have previously discovered that SV40 small t antigen trans-activates the E2A promoter during transient transfection with plasmid DNA and that this requires the EIIF sites. Small t does not bind Rb and cyclin A. Therefore, activation of EIIF-dependent transcription is likely to involve novel, undiscovered mechanisms. The transcription factors, TFIIIB and TFIIIC, control a class of RNA polymerase III-transcribed genes that include tRNA genes and the Adenovirus VA-I promoter. These transcription factors also appear to be activated in growing, transformed, or virus infected cells. Small t trans-activates the VA-I promoter during transient transfection. We have found by testing small t mutants that different structures on small t appear to be required to activate the E2A and the VA-I promoters. Therefore, there may be at least two cellular processes that are stimulated by small t. We propose to investigate the mechanisms by which these two classes of genes are regulated. We will use SV40 small t as an agent to investigate these processes. We will examine these factors by gel shift analysis using extracts of cells that express small t compared to those that do not. Where possible, we will attempt to correlate any changes detectable by gel shift analysis with increased transcription in vitro. We will continue to study small t mutants to identify the structures required for transcription regulation. This information will be used to determine how small t interacts with cellular proteins the mediate its effects off transcription. We will also examine whether the one identified cellular protein that binds to small t, the enzyme type 2A phosphatase (PP2A), mediates trans-activation by small t. Since the EIIF and TFIIIC factors appear to be regulated by phosphorylation, it is possible that small t activates transcription by inhibiting the dephosphorylation of its target transcription factors. These experiments will outline pathways of eukaryotic transcriptional control that may result in neoplastic transformation which have not been revealed by investigation of other systems.
