GENE-SPECIFIC TRANSCRIPTIONAL ACTIVITY OF THE INSULIN CAMP-RESPONSIVEELEMENT IS CONFERRED BY NF-Y IN COMBINATION WITH CAMP RESPONSE ELEMENT-BINDING PROTEIN

Cyclic AMP stimulates insulin gene transcription through a cAMP respon se element (CRE). In the present study the insulin CRE-binding protein s and their functions were investigated. A mutational analysis of nucl ear protein binding in electrophoretic mobility shift assays in combin ation with specific antisera showed that in the CRE of the rat insulin I gene the imperfect CRE octamer-like sequence TGACGTCC interacts wea kly with CREB and overlaps with two sequence motifs (TTGTTGAC and CCAA T) that bind winged helix-like proteins and the transcription factor N F-Y, respectively. Transient transfection of wild-type and mutant insu lin CRE-reporter fusion genes and the inactivation of cellular CREB or NF-Y by overexpression of the dominant negative mutants KCREB or NF-Y A29, respectively, indicate that cAMP inducibility of the insulin CRE is mediated by CREB or closely related proteins; however, NF-Y binding to the insulin CRE confers constitutive, basal activity and decreases the ability of CREB to mediate cAMP-stimulated transcription and calc ium responsiveness. Results from these studies demonstrate that NF-Y b inds to the insulin CRE and modulates the function of CREB. Together w ith the nonpalindromic sequence of the CRE octamer motif, the interact ion of NF-Y with CREB may be responsible for the gene-specific transcr iptional activity of the insulin CRE and explain why it has considerab le basal activity but is less responsive to cAMP stimulation than othe rs.