Cloning and tissue-specific functional characterization of the promoter of the rat diazepam binding inhibitor, a peptide with multiple biological actions.
Diazepam binding inhibitor (DBI) is a 10-kDa polypeptide that regulates mitochondrial steroidogenesis, glucose-induced insulin secretion, metabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABAA receptors. To investigate the regulation of DBI gene expression, three positive clones were isolated from a rat genomic library. One of them contained a DBI genomic DNA fragment encompassing 4 kb of the 5' untranslated region, the first two exons, and part of the second intron of the DBI gene. Two other overlapping clones contained a processed DBI pseudogene. Several transcription initiation sites were detected by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription startpoint usage. Transient expression experiments using DNA fragments of different length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequence, whereas full activation was achieved with 423 bp of the 5' untranslated region. DNase I protection experiments with liver nuclear proteins demonstrated three protected regions at nt -387 to -333, -295 to -271, and -176 to -139 relative to the ATG initiation codon; in other tissues the pattern of protection was different. In gel shift assays the most proximal region (-176 to -139) was found to bind several general transcription factors as well as cell type-restricted nuclear proteins which may be related to specific regulatory patterns in different tissues. Thus, the DBI gene possesses some features of a housekeeping gene but also includes a variable regulation which appears to change with the function that it subserves in different cell types.
Kolmer
M
M
Department of Biomedical Sciences, University of Tampere, Finland.
Alho
H
H
Costa
E
E
Pani
L
L
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D003850:Deoxyribonuclease I; D026261:Diazepam Binding Inhibitor; D005091:Exons; D015698:Genomic Library; D007438:Introns; D007515:Islets of Langerhans / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011544:Pseudogenes; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D003850:Deoxyribonuclease I