CLONING OF THE HUMAN 1-ALPHA,25-DIHYDROXYVITAMIN-D-3 24-HYDROXYLASE GENE PROMOTER AND IDENTIFICATION OF 2 VITAMIN-D-RESPONSIVE ELEMENTS
CLONING OF THE HUMAN 1-ALPHA,25-DIHYDROXYVITAMIN-D-3 24-HYDROXYLASE GENE PROMOTER AND IDENTIFICATION OF 2 VITAMIN-D-RESPONSIVE ELEMENTS
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DOI：
10.1016/0167-4781(95)00060-t
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发表时间：
1995-07-25
期刊：
影响因子：
--
通讯作者：
DELUCA, HF
中科院分区：
文献类型：
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作者：
CHEN, KS;DELUCA, HF
从人类 20 号染色体文库中分离出 1 α,25-二羟基维生素 D-3 (1,25-(OH)(2)D-3) 24-羟化酶的基因组 DNA 克隆。它跨度为 2.42 kb，包含前两个外显子、第二个内含子的第一个和部分，以及 1.26 kb 5' 侧翼区域。整个 5' 侧翼区域均显示出假定的转录顺式元件，包括 TATA 盒、CAAT 盒、GC 盒、维生素 D 响应元件 (VDRE)、AP1 和 AP2 位点。在 CAT 报告基因表达测定中，24-羟化酶启动子及其 1.2 kb 5' 侧翼序列可引发 1,25-(OH)(2)D-3 诱导的反式激活活性。这些假定的 DRE 的凝胶迁移率变化分析表明，两种不同的元素可以与猪肠核提取物 (PINE) 形成特定的复合物。 VDRE-PINE 复合物的特异性通过使用 VDR 特异性单克隆抗体 VXIE10B6 的 Supershift 测定进行验证。近端元件 VDRE(p) (- 172/ - 143) 由三个反义方向的直接重复半位点 GAGTCAgcgAGGTGAgcgAGGGCG 组成。远端元件 VDRE(d) (- 293/ - 273) 由两个直接重复半位点 GCGTTCaccGGGTGT 组成，也处于反义方向。两种 VDRE 都可以使用异源单纯疱疹病毒胸苷激酶 (TK) 启动子以 1,25-(OH)(2)D-3 依赖性方式指导报告基因表达。对具有天然或 TK 启动子的各种构建体中的这些 VDRE 的进一步表征表明，这两种 VDRE 都是 1,25-(OH)(2)D-3 最佳诱导 24-羟化酶表达所必需的。
A genomic DNA clone for 1 alpha,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) 24-hydroxylase was isolated from a human chromosome 20 library. It spans 2.42 kb, containing the first two exons, the first and part of the second introns, and a 1.26 kb 5'-flanking region. Putative transcription cis-elements were revealed throughout the 5'-flanking region, including TATA box, CAAT box, GC boxes, vitamin D-responsive elements (VDRE), AP1, and AP2 sites. In a CAT reporter gene expression assay, the 24-hydroxylase promoter with its 1.2 kb 5'-flanking sequence elicits a 1,25-(OH)(2)D-3-induced transactivation activity. Gel mobility shift assays of those putative DREs have identified that two different elements can form specific complexes with porcine intestinal nuclear extract (PINE). The specificity of VDRE-PINE complexes was verified by supershift assay with VDR-specific monoclonal antibody VXIE10B6. The proximal element VDRE(p) (- 172/ - 143)consists of three direct repeat half-sites, GAGTCAgcgAGGTGAgcgAGGGCG, in anti-sense orientation. The distal element VDRE(d) (- 293/ - 273) consists of two direct repeat half-sites, GCGTTCaccGGGTGT, also in anti-sense orientation. Both VDREs can direct a reporter gene expression using a heterologous herpes simplex virus thymidine kinase (TK) promoter in a 1,25-(OH)(2)D-3-dependent fashion. Further characterization of these VDREs in various constructs with either a native or TK promoter suggests that both VDREs are required for the optimal induction of 24-hydroxylase expression by 1,25-(OH)(2)D-3.