Transcriptional Regulation of the Human Cholecystokinin Gene: Composite Action of Upstream Stimulatory Factor,Sp1,and Members of the CREB/ATF-AP-1 Family of Transcription Factors

We have examined cis-elements and trans-acting factors that regulate transcription of the human cholecystokinin (CCK) gene. Transient expression of CCK promoter deletion constructs in human SK-N-MC neuroblastoma cells depicted positive cis-elements between the positions −100 to −92, −84 to −74, and −58 to −37, 5′ to the transcription initiation site. Correspondingly, DNase I protection analysis showed that trans-acting factors bound to elements within these regions. The sequences encompass a putative basic helix–loop–helix leucine zipper (bHLH-ZIP) element, an Sp1 element, and a combined cAMP- and TPA-responsive element (CRE/TRE) at positions −97 to −92, −39 to −34, and −80 to −73, respectively. Mobility and supershift assays demonstrated that upstream stimulatory factor (USF) and Sp1 bind to the former elements and competition experiments confirmed that CREB/ATF and AP-1 bind to the CRE/TRE element. Mutation of the bHLH-ZIP and CRE/TRE elements decreased the activity of the promoter by 65% and 42%, respectively. The activity of the promoter was increased six- and two-fold after stimulation with forskolin and TPA, respectively. Stimulation was eliminated after mutation of the CRE/TRE element. Co-transfection experiments with pRSV-c-jun, pSV-fos, and pRC-RSV-CREB constructs showed that jun, CREB, and AP-1 stimulate transcription. We conclude that USF, Sp1, and members of the CREB/ATF and AP-1 family of transcription factors are the major determinants of CCK gene transcription.