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Abstract
The mouse steroid 16 alpha-hydroxylase (C-P450(16 alpha)) family consists of structurally similar genes with very divergent expressions (Wong, G., Itakura, T., Kawajiri, K., Skow, L., and Negishi, M. (1989) J. Biol. Chem. 264, 2920-2927). Of five family members, the C-P450(16 alpha) gene exhibits a male-specific expression whereas the P450cb gene is equally expressed in both males and females although the level of expression is much lower than that of the C-P450(16 alpha) gene. By DNase I footprinting and in vitro transcription of the C-P450(16 alpha) gene, we determined two cis-acting transcription elements. SDI (sex difference information) in the -84/-102 region from the transcription start site is one of the two cis-acting transcription elements, which is specific to the sex-specific C-P450(16 alpha) gene and confers a high transcription level of this gene. We substituted the nucleotide(s) in the SDI sequence to the corresponding base(s) in the P450cb gene promoter and tested each substituted gene promoter by in vitro transcription. The results indicate that the function of SDI as the transcription element depends most critically on the nucleotides at positions -100 to -98. Another cis-acting transcription element, CTE (common transcription element), is located between -44 and -68 in both C-P450(16 alpha) and P450cb genes. A column chromatography of nuclear extract indicates that the proteins bound to SDI and CTE are different. We speculate therefore that SDI is a specific and necessary element involved in the male-specific C-P450(16 alpha) gene transcription. The sequence comparisons of the corresponding regions of SDI and CTE among the mouse and the closely related rat P450 genes suggest that SDI is a newly evolved cis-acting element in the sex-specific C-P450(16 alpha) gene which arose through gene duplication and selective nucleotide substitution.
Highlights
C-P4501e, gene exhibits a male-specific expression whereas the P450cb gene is expressed in both males and females the level of expression is much lower than that of the C-P4501e, gene
In order to find the mechanism by which sex-dependent P450 gene transcription is regulated, we examine here, by DNase I footprinting and in vitro transcription, the functional diversity of the cis-acting transcription elements in the C-P45016arand P450cb genes
This paper identified two adjacent cis-acting transcription elements in the 5’-flanking regions of the P450 genes within the mouse C-P45016, family
Summary
We used PCR to synthesize the C-P450i6, gene promoters whose nucleotides in the SD1 sequence were mutated to the counterparts in the P450cb gene. For this purpose, either the -117/-16 or a -HO/. The end-labeled DNA (1 x lo cpm) was incubated with 5 or 10 pg of the nuclear proteins in 10 mM Tris-HCl buffer (pH 7.5) containing 2.5 fig of poly(dI.dC), 50 mM NaCI, 2.5 mM MgCl,, 1.0 mM dithiothreitol, 0.5 mM EDTA, and 5% glycerol. The digested DNAs were purified by phenol-chloroform extractions, precipitated with ethanol, dried, and dissolved in 4 ~1 of 90% formamide containing 0.5 X TBE, 0.01%.
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