A Downstream Intergenic Cluster of Regulatory Enhancers Contributes to the Induction of CYP24A1 Expression by 1α,25-Dihydroxyvitamin D3
A Downstream Intergenic Cluster of Regulatory Enhancers Contributes to the Induction of CYP24A1 Expression by 1α,25-Dihydroxyvitamin D3
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DOI：
10.1074/jbc.m110.119958
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发表时间：
2010-05-14
影响因子：
4.8
通讯作者：
Pike, J. Wesley
中科院分区：
文献类型：
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作者：
Meyer, Mark B.;Goetsch, Paul D.;Pike, J. Wesley
CYP 24 A1的表达被1，25-二羟基维生素D-3（1，25（OH）（2）D-3）通过维生素D受体（VDR）/类维生素A X受体（RXR）异二聚体上调，该异二聚体与位于近端启动子附近的两个维生素D反应元件（VDRE）结合。有趣的是，尽管1，25（OH）（2）D-3诱导VDR/RXR与含VDRE的近端启动子结合，但VDR/RXR异二聚体也定位于位于人CYP 24 A1基因下游50-69 kb和小鼠Cyp 24 a1基因下游35-45 kb的基因间区域中的至少4个潜在增强子的簇，如ChIP芯片和ChIP-seq分析所示。为了解决该下游区域和潜在的VDRE是否位于介导的CYP 24 A1诱导内，我们构建了重组野生型和突变型细菌人工染色体克隆，其跨越小鼠和人类基因座，并含有插入其3 '-非翻译区的荧光素酶报告基因。这些克隆在稳定转染细胞中的活性表明，近端和推定的下游元件均有助于1，25（OH）（2）D-3上调CYP 24 A1。使用转染的增强子片段的进一步分析导致在这些下游区域中的几个中鉴定出起作用的调控元件。使用ChIP芯片分析的辅助调节剂招聘的其他研究揭示了位于近端和位于启动子下游的区域之间的相似性和差异。这些辅助调节因子的募集可能是RNA聚合酶II和组蛋白H4乙酰化增加的原因，这也是在整个基因座的增强子位点上对1，25（OH）（2）D-3的反应中观察到的。我们的结论是，一个更复杂的机制是负责在靶细胞中的维生素D激素诱导的CYP 24 A1的显着上调。
CYP24A1 expression is up-regulated by 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) via a vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer that binds to two vitamin D response elements (VDREs) located near the proximal promoter. Interestingly, although 1,25(OH)(2)D-3 induced VDR/RXR binding to the VDRE-containing proximal promoter, the VDR/RXR heterodimer also localized to a cluster of at least four potential enhancers located in intergenic regions 50-69 kb downstream of the human CYP24A1 gene and 35-45 kb downstream of the mouse Cyp24a1 gene as revealed by ChIP-chip and ChIP-seq analyses. To address whether this downstream region and potential VDREs located within mediated CYP24A1 induction, we constructed recombinant wild-type and mutant bacterial artificial chromosome clones that spanned mouse and human loci and contained luciferase reporters inserted into their 3'-untranslated regions. The activity of these clones in stably transfected cells revealed that both the proximal and the putative downstream elements contributed to CYP24A1 up-regulation by 1,25(OH)(2)D-3. Further analysis using transfected enhancer fragments led to the identification of contributing regulatory elements in several of these downstream regions. Additional studies of coregulator recruitment using ChIP-chip analysis revealed both similarities and differences between the region located proximal to and those located downstream of the promoter. Recruitment of these coregulators was likely responsible for the increase in RNA polymerase II and histone H4 acetylation, which was also observed in response to 1,25(OH)(2)D-3 at the enhancer sites across the locus. We conclude that a more complex mechanism is responsible for the striking CYP24A1 up-regulation induced by the vitamin D hormone in target cells.