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 regulate s mitochondrial steroidogenesis, glucose-induced insulin secretion, me tabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABA(A) receptors. To investigate the regulation of DBI gene expressio n, 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 i ntron of the DBI gene. Two other overlapping clones contained a proces sed DBI pseudogene. Several transcription initiation sites were detect ed by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription start point usage. Transient expression experiments using DNA fragments of d ifferent length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequ ence, whereas full activation was achieved with 423 bp of the 5' untra nslated region. DNase I protection experiments with fiver nuclear prot eins 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 t he most proximal region (-176 to -139) was found to bind several gener al transcription factors as well as cell type-restricted nuclear prote ins 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 w ith the function that it subserves in different cell types.