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.
Highlights
Crease in gene transcription [6]
The same mutations were introduced into reporter gene constructs in a pGCAT-A-based vector containing 1400 bp of 5Ј MUC1 flanking sequence (Ϫ1400/ϩ33) and transiently transfected cells were examined for expression of the CAT gene
In this report we have focused on the analysis of two Sp1 sites in the MUC1 promoter located in regions previously characterized as being functionally important at Ϫ576/Ϫ567 and Ϫ99/Ϫ90, respectively
Summary
Crease in gene transcription [6]. tissue specificity of expression is seen at the level of mRNA. An analysis of the elements of the MUC1 promoter and the factors interacting with these elements that play a role in regulation of expression of MUC1 in epithelial cells and in tumors is of interest. In vitro functional studies have identified a minimal promoter of around 600 bp upstream from the transcripbinding activity in nuclear extracts correlated with tion starting site [9, 10] These studies showed that the most both promoter activity and the levels of endogenous important regions controlling transcription in breast cancer transcription in different breast cancer cell lines. Pressing cells since they direct tissue-specific expression when coupled to a foreign enhancer These results indicate that the regulation of MUC1 expression is controlled by several transactivating factors
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