REPRESSION OF VASOPRESSIN GENE-EXPRESSION BY GLUCOCORTICOIDS IN TRANSGENIC MICE - EVIDENCE OF A DIRECT MECHANISM MEDIATED BY PROXIMAL 5'-FLANKING SEQUENCE

Glucocorticoids are known to exert multiple effects upon neuronal syst ems and neuronal gene expression but the molecular mechanisms through which these effects are mediated are largely undefined. In this study, a transgenic mouse model that expresses a bovine vasopressin transgen e was used to investigate the mechanisms by which this neuropeptide ge ne is repressed by glucocorticoids. Using both northern analysis and a reverse transcriptase-polymerase chain reaction assay, depletion of g lucocorticoids with the 11,beta-hydroxylase inhibitor metyrapone was s hown to result, in a dexamethasone-reversed increase in ectopic adrena l transgene messenger RNA revels. This result shows that sequences wit hin the confines of the 3.5 kb transgene are sufficient to mediate rep ression by glucocorticoids, and indicates the involvement of a type II glucocorticoid receptor mechanism which is independent of cellular co ntext. Evidence for the involvement of cis-acting repressive elements in the proximal 5' flanking sequence was obtained in further studies i n which bovine transgene constructs were shown to be negatively regula ted by dexamethasone in 293 cells. The further demonstration that reco mbinant glucocorticoid receptor binds to a vasopressin promoter fragme nt in an in vitro electrophoretic mobility shift assay provided additi onal evidence of a direct mechanism of repression. Both in vitro studi es were consistent with the presence of a glucocorticoid regulatory el ement within the region -300 to -155 of the transcription start site. The use of an in vivo transgenic system combined with in vitro analyse s of gene promoter fragments enabled the characterization of the molec ular mechanisms which effect physiological changes in vasopressin gene expression, and provided evidence of a direct mechanism of repression mediated by sequences within the vasopressin gene promoter. (C) 1997 IBRO. Published by Elsevier Science Ltd.