Two GC boxes (Sp1 sites) are involved in regulation of the activity of the epithelium-specific MUC1 promoter

Abstract

In this report, we have analyzed the function of two Sp1 sites present in the epithelium-specific MUC1 promoter. Using promoter-reporter gene (CAT) constructs, we found that mutagenesis of either of the Sp1 binding motifs at -576/-568 and -99/-90, reduced transcription in MUC1-expressing epithelial cell lines. However, abolition of the binding site at -99/-91 by mutagenesis also resulted in increased transcriptional activity in non-epithelial cell lines, suggesting involvement of the site in tissue-specific expression. In vitro binding assays revealed a novel binding motif at -101/-89 (AGGGGGCGGGGTT), which overlaps but differs from the Sp1 consensus motif by having an adenine residue in the 5'-flanking sequence. The 5'-flanking sequence appeared to be important for binding of an Sp1-unrelated factor (SpA) but not for binding of Sp1. Site-directed mutagenesis of the motif into a site able to bind Sp1, but unable to bind SpA, resulted in an increased level of transcription of the CAT reporter gene in all cell lines tested, suggesting a repressive effect of the novel factor on transcription. The ratio between the Sp1 and SpA binding activity in nuclear extracts correlated with both promoter activity and the levels of endogenous transcription in different breast cancer cell lines. Our results are consistent with the idea that the relative activities of the two factors may be involved in the up- regulation of expression of the MUC1 gene seen in breast and other carcinomas.