Cloning and characterization of the 5 ' flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells

Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consi sting of a mucin subunit ascites sialoglycoprotein-l (ASGP-1) and a transme mbrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide: precursor, and further processed to yield the ma ture ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mech anisms. A crude exon/intron map of the 5' regions of the SMC/Muc4 genes gen erated from clones isolated from a normal rat liver genomic DNA library rev eals that this gene has a small first exon comprising the 5' untranslated r egion and signal peptide, followed by a large intron. The second exon appea rs to be large, comprising the 5' unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to t hat reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb o f the 5'-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue -specific promoters. Reporter constructs generated from this 2.4 kb fragmen t demonstrate promoter activity in primary rat mammary epithelial cells (ME C), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell spec ificity. Deletion constructs of this sequence transfected into rat MEC or H CT-116 cells demonstrate greatly varying levels of activity, suggesting tha t there are positive and negative, as well as tissue-specific, regulatory e lements in this sequence. Taken together, these data suggest that the rat S MC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue -specific, regulatory elements in the sequence.