Miyamoto K, Kesterson R A, Yamamoto H, Taketani Y, Nishiwaki E, Tatsumi S, Inoue Y, Morita K, Takeda E, Pike J W
Department of Clinical Nutrition, Tokushima University, Japan.
Mol Endocrinol. 1997 Jul;11(8):1165-79. doi: 10.1210/mend.11.8.9951.
The vitamin D receptor (VDR) is known to mediate the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. The regulation of this ligand-activated cellular transcription factor is reported to occur at both transcriptional and posttranslational levels. To begin to address the molecular basis by which the VDR gene is regulated transcriptionally, we report here an initial characterization of the human VDR gene and its promoter. We isolated several overlapping A-phage and cosmid clones that cover more than 100 kb of human DNA and contained the entire VDR gene. The gene is comprised of 11 exons that, together with intervening introns, span approximately 75 kb. The noncoding 5'-end of the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons 2-9) encode the structural portion of the VDR gene product. While primer extension and S1 nuclease-mapping studies reveal several common transcriptional start sites, three unique mRNA species are produced as a result of the differential splicing of exons 1B and 1C. The DNA sequence lying upstream of exon 1A is GC rich and does not contain an apparent TATA box. Several potential binding sites for the transcription factor SP1 and other activators are evident. Fusion of DNA fragments containing putative promoter sequences upstream of the luciferase structural gene followed by transient transfection of these plasmids into several mammalian cell lines resulted in significant reporter activity. Due to the size and complexity of the 5'-end of the VDR gene, we examined the activity of a DNA fragment surrounding exon 1C. An intron fragment 3' of exon 1C conferred retinoic acid responsivity when fused to a reporter gene plasmid, suggesting a molecular mechanism for the previously observed ability of retinoic acid to induce the VDR. The recovery of the gene for the human VDR will enable further studies on the transcriptional regulation of this gene.
维生素D受体（VDR）通过调节靶基因表达的能力介导1,25 - 二羟基维生素D3的多效生物学作用。据报道，这种配体激活的细胞转录因子的调节发生在转录和翻译后水平。为了开始探讨VDR基因转录调控的分子基础，我们在此报告人类VDR基因及其启动子的初步特征。我们分离了几个重叠的λ噬菌体和粘粒克隆，它们覆盖了超过100 kb的人类DNA，并包含整个VDR基因。该基因由11个外显子组成，外显子与中间的内含子一起跨越约75 kb。基因的非编码5'端包括外显子1A、1B和1C。另外八个外显子（外显子2 - 9）编码VDR基因产物的结构部分。虽然引物延伸和S1核酸酶图谱分析揭示了几个常见的转录起始位点，但由于外显子1B和1C的差异剪接产生了三种独特的mRNA种类。外显子1A上游的DNA序列富含GC，且不包含明显的TATA盒。转录因子SP1和其他激活剂的几个潜在结合位点很明显。将含有荧光素酶结构基因上游推定启动子序列的DNA片段融合，然后将这些质粒瞬时转染到几种哺乳动物细胞系中，导致显著的报告基因活性。由于VDR基因5'端的大小和复杂性，我们检查了围绕外显子1C的DNA片段的活性。外显子1C 3'端的一个内含子片段与报告基因质粒融合时赋予视黄酸反应性，这表明了视黄酸先前观察到的诱导VDR能力的分子机制。人类VDR基因的获得将使对该基因转录调控的进一步研究成为可能。
