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.
Interleukin-1 beta (IL-1beta) is one of the most important inflammatory mediators in human inflammatory and immunological diseases. The regulation of human IL-1beta gene expression has been studied for several years, and a few regulatory elements have been discovered in the promoter region. However, little is known about negative regulation of IL-1beta expression at the transcriptional level, which may play an important role in anti-inflammatory and immunosuppressive effects. We have identified a negative regulatory element located in the region between -685 and -395. Within this region, a 19-bp nuclear factor binding site (-570 to -552) was characterized by DNase I footprinting and electromobility shift assay. A consensus sequence for a negative glucocorticoid response element (nGRE) and a transcription activator protein-2 binding site were noted within this footprint. Functional studies showed a 2.5-fold increase in promoter activity when this 19-bp binding site was deleted in the reporter constructs IL-1beta/CAT and IL-1beta/SV40 promoter/CAT. Dexamethasone (10(-8) M) repressed chloramphenicol acetyltransferase (CAT) production by 75% in the wild-type fragment but not in a deletion mutant lacking the 19-bp site. A protein of about 150 kD that bound to this negative regulatory sequence was identified by UV cross-linking. This is the first description of a negative regulatory region responsive to glucocorticoids in a cytokine gene.
The biosynthesis of glucocorticoids and mineralocorticoids requires isozymes of P450c11. Two human isozymes are known: P450c11 beta, encoded by the CYP11B1 gene, has 11 beta-hydroxylase activity; P450c11AS, encoded by the CYP11B2 gene, has 11 beta-hydroxylase, 18-hydroxylase, and aldosterone synthase activities. Recent data show that the rat genome has four CYP11B genes, three of which are functional, and one of which has novel behaviors. As the number of human CYP11B genes was unknown and as the existence of novel P450c11 isozymes might have implications in the study of hypertension, we sought to determine if the human genome, like the rat genome, contained more than two CYP11B genes. Southern blotting of human genomic DNA digested with StuI suggested the existence of at least four human CYP11B genes. Similar analysis of cosmid clones suggested multiple CYP11B genes. However, cloning and sequencing of the multiple hybridizing fragments showed that there are only two CYP11B genes in the human genome, and that the "extra" bands seen were due to spurious hybridization. The absence of additional CYP11B genes in the human genome analogous to those in the rat narrows the search for genes that contribute to low renin hypertension.
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