Zella Lee A, Kim Sungtae, Shevde Nirupama K, Pike J Wesley
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Mol Endocrinol. 2006 Jun;20(6):1231-47. doi: 10.1210/me.2006-0015. Epub 2006 Feb 23.
The biological actions of 1,25-(OH)2D3 are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)2D3 is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)2D3 to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation. After establishing the ability of 1,25-(OH)2D3 to stimulate VDR mRNA up-regulation both in bone in vivo and in osteoblastic cells, we screened the mouse VDR gene locus from 20 kb upstream of the gene's transcriptional start site (TSS) to 10 kb downstream of the final exon to identify VDR binding sites using chromatin immunoprecipitation-DNA microarray (ChIP-chip) analysis. Three conserved regions were identified 20, 27, and 29 kb downstream of the TSS. VDR binding to these sites in response to 1,25-(OH)2D3 was confirmed by ChIP analysis and was accompanied by differential localization of retinoid X receptor, histone acetylation, and RNA polymerase II recruitment. One of these regions was able to confer 1,25-(OH)2D3 regulation to downstream promoters, thereby permitting identification and characterization of the regulatory element located within. Importantly, a highly conserved region within the human VDR gene analogous to that discovered in the mouse was also capable of mediating 1,25-(OH)2D3 response. Our results demonstrate that 1,25-(OH)2D3 and its receptor autoregulate the expression of the VDR gene. The location of these regulatory regions and their apparent distances from the TSS are consistent with new findings suggesting the emerging relevance of distant enhancers.
1,25 -（OH）₂D₃的生物学作用是由维生素D受体（VDR）介导的，VDR是一种与靶基因结合并改变其表达的蛋白质。1,25 -（OH）₂D₃还能够诱导VDR基因自身的转录。在本研究中，我们探讨了1,25 -（OH）₂D₃在骨细胞中诱导VDR基因表达的能力以及这种上调的作用机制。在确定1,25 -（OH）₂D₃在体内骨组织和成骨细胞中均能刺激VDR mRNA上调后，我们使用染色质免疫沉淀 - DNA微阵列（ChIP - chip）分析，从基因转录起始位点（TSS）上游20 kb到最后一个外显子下游10 kb对小鼠VDR基因位点进行筛选，以鉴定VDR结合位点。在TSS下游20、27和29 kb处鉴定出三个保守区域。通过ChIP分析证实了VDR在1,25 -（OH）₂D₃作用下与这些位点的结合，同时伴有视黄酸X受体的差异定位、组蛋白乙酰化以及RNA聚合酶II的募集。其中一个区域能够将1,25 -（OH）₂D₃的调控作用赋予下游启动子，从而使得位于其中的调控元件得以鉴定和表征。重要的是，在人类VDR基因中发现的一个与小鼠中类似的高度保守区域也能够介导1,25 -（OH）₂D₃反应。我们的结果表明，1,25 -（OH）₂D₃及其受体对VDR基因的表达进行自我调节。这些调控区域的位置及其与TSS的明显距离与新发现一致，提示远距离增强子的相关性日益凸显。
