CHARACTERIZATION OF THE MOUSE EPIDERMAL GROWTH-FACTOR PROMOTER AND 5'-FLANKING REGION - ROLE FOR AN ATYPICAL TATA SEQUENCE

As a step toward delineating mechanisms that regulate its activity, we have characterized the mouse epidermal growth factor (EGF) promoter, Primer extension and S1 nuclease analyses identified prominent (+1/+2) and minor (+28) transcription start sites, with the dominant +1/+2 si te located 33 bases downstream from a TTTAAA sequence, A restriction f ragment that spanned these start sites and contained 390 base pairs of 6'-flanking sequence directed transcription from the +1/+2 site in vi tro in the presence of HeLa cell nuclear extracts. Additionally, it pr omoted expression of a coupled luciferase reporter gene in transfected cell lines, The inclusion of additional 5'-flanking sequence either s timulated or inhibited luciferase expression depending on the cell lin e. Approximately 2 kilobases of EGF 5'-flanking sequence was determine d and found to contain several motifs with partial homology to steroid hormone response elements. Despite this fact and evidence that EGF ex pression might be regulated by androgens in vitro, EGF promoter-lucife rase constructs were not steroid-responsive in cells cotransfected wit h steroid receptor expression vectors. An oligonucleotide containing t he aforementioned TTTAAA sequence specifically bound TATA-binding prot ein and TFILA in gel shift assays, and an EGF promoter-luciferase cons truct in which the core TA dinucleotide was mutated to CG was not acti ve in transfected cells. These data suggest that the TTTAAA sequence f unctions as an atypical TATA box.