Transcriptional regulatory elements of the human gene for cytochrome P450c21 (steroid 21-hydroxylase) lie within intron 35 of the linked C4B gene

The CYP21 gene, which encodes P450c21, the adrenal steroid SI-hydroxylase n eeded for glucocorticoid synthesis, lies in the major histocompatibility lo cus only 2.3 kilobase pairs (kb) downstream from the C4 gene. A 300-base pa ir (bp) proximal promoter and two upstream regions within C4 are needed for expression of mouse CYP21; the human gene also has a proximal promoter, bu t upstream elements have not been studied. To search for upstream regulator y elements in human CYP21B, we examined up to 9 kb of 5'-flanking DNA by tr ansient transfection into human adrenal NCI-H295A cells. The 300-bp proxima l promoter had substantial activity, but constructs retaining the DNA betwe en -4.6 and -5.6 kb had increased activity, indicating the presence of dist al elements. This region does not correspond to the mouse upstream regions, lying further upstream within intron 35 of C4B, which encompasses the prev iously described "Z promoter," DNase I footprinting located two elements, F 1 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 orienta tion-independent expression to a heterologous promoter. Mutations abolished formation of the DNA-protein complexes but only partially decreased expres sion. We identified a third site, F3, lying at -33 to -42 bp from Z. Compet itive gel mobility supershift assays and co-transfection studies with SF-1 produced in vitro indicate F2 and F3 bind SF-1; BLAST searches and Southwes tern blotting suggest that NF-W2 may bind F1. These results indicate that t he Z promoter is a component of the CYP21 promoter needed to drive its adre nal-specific expression and that CYP21 transcription elements within C4 hav e kept these two genes linked during evolution.