Construction of predictive promoter models on the example of antibacterial response of human epithelial cells
The initiation point for MUC2 gene transcription is located within a 7000-base GC-rich region of the mucin gene cluster found on chromosome 11p15.5. The promoter activity of the 5'-flanking region of the MUC2 gene was examined following its cloning into the luciferase-producing pGL2-Basic reporter vector. A short segment comprising bases -91 to -73 relative to the start of transcription was found to be important for basal promoter activity in all cell lines tested. Electrophoretic mobility shift assays demonstrated nuclear protein binding to this region, which contains the consensus CACCC motif (5'-GCCACACCC). This element has been shown to be functionally important in several promoters that are active in diverse cell types. Competition experiments using an Sp1 oligonucleotide and antibody supershift experiments indicated that both Sp1 and other Sp1 family members bind to this element. Inclusion of the region between bases -228 and -171 in pGL2-Basic constructs increased normalized luciferase reporter activity by almost 3-fold in C1a cells, which produce relatively high levels of MUC2 mRNA. Significantly lower levels of normalized luciferase activity resulted when the same construct was transfected into cultured cell lines that express low or undetectable levels of MUC2, suggesting a possible role for this region in conferring cell-type specificity of expression. We also demonstrate, using actinomycin D, that the MUC2 mRNA is long-lived, at least in cultured cells. Moreover, no evidence was found that the MUC2 mRNA turned over more rapidly in LS174T cells, which produce relatively low levels of MUC2 mRNA, as compared with C1a cells, which produce high levels of mRNA. Thus a long mRNA half-life appears to be an important mechanism involved in achieving elevated levels of MUC2 mRNA.