Structural and functional analysis of 5' flanking and intron 1 sequences of the hamster P-glycoprotein pgp1 and pgp2 genes

Abstract

Several studies have demonstrated that regulation of P-glycoprotein gene expression at the transcriptional level is complex and involves multiple regulatory mechanisms. To investigate the transcriptional regulation of P-glycoprotein genes, genomic DNA fragments containing the 5' end of the hamster pgp1 and pgp2 genes were isolated and characterized. The pgp1 5' flanking sequences were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and a series of 5' deletions were constructed. Transient expression of these CAT constructs into Chinese hamster ovary (CHO) cells revealed that the pgp1 promoter is regulated by multiple positive and negative regulatory elements. One particular region between -489 and -255 was shown to possess silencer activity. This region contains two putative negative elements that are also present in the silencer regions of several other genes. Intron 1 sequences of the Pgp genes were also examined and shown to be highly conserved both between family members and across species. Transient expression studies revealed that intron 1 sequences possess enhancer activity. Thus, it was demonstrated that sequences upstream and downstream of the transcriptional start site are important for the regulation of P-glycoprotein gene expression.