Tissue-Specific Transcription of P-450(11β) Gene In Vitro1

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Abstract
The transcriptional activity of P-450(11β) gene was studied with an in vitro transcription system using nuclear extracts prepared from bovine adrenal cortex. Template plasmids were constructed with the promoter region of the bovine P-450(11β) gene and a G-less cassette as a reporter gene. Deletion analysis of the promoter region revealed two neighboring sites, Ad1 and Ad2, which are necessary for efficient transcription of the templates. These sites are highly conserved among bovine, mouse, and human genes. Though the Ad1 site, TGACGTGA, showed high homology to the consensus sequence of the cAMP-responsive element, TGACGTCA, with one base substitution, the Ad2 site showed no similarity to any regulatory element reported so far. Gel shift assay using synthetic nucleotides containing Ad1 and/or Ad2 indicated that three factors bound to these regions in adrenal cortex nuclear extract, two factors to Ad2 and one factor to Ad1. DNase I footprint analysis also showed the binding of factors to the Ad1 and Ad2 regions. Catenated synthetic nucleotides containing Ad1 and/or Ad2 inhibited the transcriptional activity of P-450(11β) gene. The P-450(11β) promoter expressed a stronger transcriptional activity in the nuclear extract of adrenal cortex than in that of liver. On the other hand, the promoter of α-1-antitrypsin gene, which is expressed only in liver, showed an opposite tissue specificity of transcription. Addition of aderenal cortex nuclear extract to liver nuclear extract activated specifically the P-450(11β) promoter in proportion to the quantity of adrenal cortex nuclear extract added. The enhancing effect of the adrenal cortex nuclear extract suggested that the factors detected in the nuclear extract of adrenal cortex are necessary for the transcription of P-450(11β) genes.