Gene-specific transcriptional activity of the insulin cAMP-responsive element is conferred by NF-Y in combination with cAMP response element-binding protein.
Cyclic AMP stimulates insulin gene transcription through a cAMP response element (CRE). In the present study the insulin CRE-binding proteins and their functions were investigated. A mutational analysis of nuclear protein binding in electrophoretic mobility shift assays in combination with specific antisera showed that in the CRE of the rat insulin I gene the imperfect CRE octamer-like sequence TGACGTCC interacts weakly with CREB and overlaps with two sequence motifs (TTGTTGAC and CCAAT) that bind winged helix-like proteins and the transcription factor NF-Y, respectively. Transient transfection of wild-type and mutant insulin CRE-reporter fusion genes and the inactivation of cellular CREB or NF-Y by overexpression of the dominant negative mutants KCREB or NF-YA29, 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 calcium responsiveness. Results from these studies demonstrate that NF-Y binds to the insulin CRE and modulates the function of CREB. Together with the nonpalindromic sequence of the CRE octamer motif, the interaction of NF-Y with CREB may be responsible for the gene-specific transcriptional activity of the insulin CRE and explain why it has considerable basal activity but is less responsive to cAMP stimulation than others.
Eggers
A
A
Department of Molecular Pharmacology, University of Göttingen, D-37070 Göttingen, Germany.
Siemann
G
G
Blume
R
R
Knepel
W
W
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D018257:Helix-Loop-Helix Motifs; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; C513169:Foxm1 protein, rat; D007328:Insulin; D009841:Oligonucleotides; D014157:Transcription Factors; C484091:insulin response element binding protein, rat