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 site located 33 bases downstream from a TTTAAA sequence. A restriction fragment that spanned these start sites and contained 390 base pairs of 5-flanking sequence directed transcription from the ؉1/؉2 site in vitro in the presence of HeLa cell nuclear extracts. Additionally, it promoted expression of a coupled luciferase reporter gene in transfected cell lines. The inclusion of additional 5-flanking sequence either stimulated or inhibited luciferase expression depending on the cell line. Approximately 2 kilobases of EGF 5-flanking sequence was determined and found to contain several motifs with partial homology to steroid hormone response elements. Despite this fact and evidence that EGF expression might be regulated by androgens in vivo, EGF promoter-luciferase constructs were not steroid-responsive in cells cotransfected with steroid receptor expression vectors. An oligonucleotide containing the aforementioned TTTAAA sequence specifically bound TATA-binding protein and TFIIA in gel shift assays, and an EGF promoter-luciferase construct in which the core TA dinucleotide was mutated to CG was not active in transfected cells. These data suggest that the TTTAAA sequence functions as an atypical TATA box.

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