PROTEIN INTERACTIONS AT SP1-LIKE SITES IN THE TGF-ALPHA PROMOTER AS VISUALIZED BY IN-VIVO GENOMIC FOOTPRINTING

Transcription from the rat TGP alpha promoter initiates at two predomi nant sites (-188 and -58) in a G+C-rich region that does not contain T ATA or CAAT motifs. Previous studies using transfected reporter constr ucts implicated the transcription factor Sp1 in active expression from the promoter, particularly from the -58 site (Chen et al., 1992; Shin et al., 1992). In the present report we have examined the functionali ty of two adjacent clusters of Sp1-like recognition sites that are loc ated in the upstream portion of the promoter from -300 to -273. A doub le-stranded oligonucleotide, which spanned this region and contained t he putative Sp1 elements, demonstrated similar gel-mobility shifts in the presence of both crude HeLa cells nuclear extract and pure Sp1 pro tein. Mutations that simultaneously altered several of the overlapping Sp1 elements significantly reduced the gel-mobility shift activity of this oligonncleotide probe and, when introduced into the promoter tem plates, inhibited transcription in vitro from the proximal -188 start site. To confirm the binding of protein to these sites in cells, we ca rried out an in vivo genomic footprinting analysis of this portion of the TGF alpha promoter in normal and transformed rat liver epithelial cell lines that express the endogenous gene at varying levels. This an alysis revealed clear evidence of protein/DNA interaction at Sp1-like sites in the -300 and -273 region in cells actively expressing the gen e but not in a normal, parental cell. line that expressed very low lev els of TGF alpha mRNA. Collectively, these results corroborate the fun ctional importance of Sp1 binding elements in the -300 to -273 region, and together with previous findings, indicate that two clusters of Sp 1 binding sites respectively determine levels of transcription from th e -188 and -58 start sites. Our additional finding that Sp1 mRNA and p rotein were present at similar levels in normal and transformed cells that expressed the endogenous TGF alpha gene at markedly different lev els, suggests that the activity of the TGF alpha promoter could be reg ulated via the accessibility of Sp1 protein.