Transcriptional Regulatory Elements of the Human Gene for Cytochrome P450c21 (Steroid 21-Hydroxylase) Lie within Intron 35 of the Linked C4B Gene

• Published in: The Journal of biological chemistry 1999-12, Vol.274 (53), p.38097-38106
• Main Authors: Wijesuriya, S D, Zhang, G, Dardis, A, Miller, W L
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; CYP21B gene; DNA Primers; Gene Expression Regulation, Enzymologic - genetics; Genes, Reporter; Humans; Introns - genetics; Mice; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Steroid 21-hydroxylase; Steroid 21-Hydroxylase - genetics; Transcription, Genetic - genetics; Transfection; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.53.38097

The CYP21 gene, which encodes P450c21, the adrenal steroid 21-hydroxylase needed for glucocorticoid synthesis, lies in the major histocompatibility locus only 2.3 kilobase pairs (kb) downstream from the C4 gene. A 300-base pair (bp) proximal promoter and two upstream regions within C4 are needed for expression of mouse CYP21 ; the human gene also has a proximal promoter, but upstream elements have not been studied. To search for upstream regulatory elements in human CYP21B , we examined up to 9 kb of 5′-flanking DNA by transient transfection into human adrenal NCI-H295A cells. The 300-bp proximal promoter had substantial activity, but constructs retaining the DNA between −4.6 and −5.6 kb had increased activity, indicating the presence of distal elements. This region does not correspond to the mouse upstream regions, lying further upstream within intron 35 of C4B , which encompasses the previously described “Z promoter.” DNase I footprinting located two elements, F1 and F2, lying −186 to −195 bp and −142 to −151 bp upstream from the Z cap site (−4862 to −4871 and −4818 to −4827 bp upstream of the CYP21B cap site). Each element formed a specific DNA-protein complex and conferred orientation-independent expression to a heterologous promoter. Mutations abolished formation of the DNA-protein complexes but only partially decreased expression. We identified a third site, F3, lying at −33 to −42 bp from Z. Competitive gel mobility supershift assays and co-transfection studies with SF-1 produced in vitro indicate F2 and F3 bind SF-1; BLAST searches and Southwestern blotting suggest that NF-W2 may bind F1. These results indicate that the Z promoter is a component of the CYP21 promoter needed to drive its adrenal-specific expression and that CYP21 transcription elements within C4 have kept these two genes linked during evolution.