Functional characterization of a cAMP-responsive element of the rat insulin I gene.
Analogues of cAMP have been reported to increase insulin mRNA levels in normal rat beta-cells and hamster insulinoma cells (HIT). To define the mechanisms by which cAMP modulates insulin gene expression, we first investigated its effects on the transcriptional rate of the insulin gene in HIT cells. Nuclear run-on assays revealed a 4-fold increase in transcription observed as early as 1 h after stimulation. To characterize the cis-acting sequences of the rat insulin I gene promoter and the trans-acting factors mediating the cAMP effect on insulin gene transcription, we constructed DNA plasmids containing various lengths of the rat insulin I gene 5'-flanking region linked to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Studies of the transcriptional activity of 5'-deletionally and pointly mutated plasmids after transfection into HIT cells revealed the presence of a cAMP-responsive element (CRE), TGACGTCC, between -177 and -184 relative to the transcriptional start site, whose sequence closely matches the previously defined CREs, present in cAMP-responsive genes. Gel retardation and Southwestern assays identify a protein of molecular weight approximately 43,000, binding specifically to the insulin CRE. We conclude that the rat insulin I gene is regulated by cAMP through a CRE and that the nuclear protein interacting with it might be similar or identical to the previously purified cAMP-responsive protein, CREB.
Philippe
J
J
Department of Microbiology, Centre Médical Universitaire, Geneva, Switzerland.
Missotten
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D007328:Insulin / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013045:Species Specificity; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000242:Cyclic AMP