Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.
Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.
