Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP

Abstract

Transcription of the vasoactive intestinal polypeptide (VIP) gene is regulated by cAMP. To identify the nucleotide sequences in the human VIP gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human VIP gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat pheochromocytoma cell line PC12 were assayed by measuring CAT activity in the cell lysates. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of VIP-CAT fusion genes. Deletional analysis demonstrated that a region between -86 and -70 nucleotides upstream from the transcriptional origin of the human VIP gene was responsible for stimulation by forskolin. This region was able to confer cAMP-responsiveness to a gene that is not normally regulated by cAMP. Two copies of a 5 base pair motif, 5'-CGTCA-3', are required for activity of the VIP cAMP regulatory region. This motif is also present in the cAMP regulatory region of several other eukaryotic genes.