Loudig O, Babichuk C, White J, Abu-Abed S, Mueller C, Petkovich M
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Mol Endocrinol. 2000 Sep;14(9):1483-97. doi: 10.1210/mend.14.9.0518.
The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of murine retinoic acid receptor-gamma (RARgamma) and retinoid X receptor-alpha (RXRalpha) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).
维甲酸（RA）的分解代谢是限制特定组织和细胞暴露于RA的重要机制。我们最近报道了在斑马鱼、小鼠和人类中鉴定出一种RA诱导的细胞色素P450 [P450RAI（CYP26）]，它被证明负责RA的分解代谢。P450RAI在发育过程中表现出复杂的时空表达模式，并且在某些组织和细胞系中可被外源性RA处理高度诱导。对P450RAI启动子近端上游区域的序列分析显示，斑马鱼、小鼠和人类之间具有高度保守性。该启动子区域包含一个典型的视黄酸反应元件（5'-AGT-TCA-(n)5-AGTTCA-3'），嵌入在一个32bp的区域（称为R1）内，在所有三个物种中都是保守的。使用该元件进行的电泳迁移率变动分析证明了小鼠视黄酸受体-γ（RARγ）和类视黄醇X受体-α（RXRα）蛋白的特异性结合。将小鼠P450RAI启动子与荧光素酶报告基因融合的瞬时转染实验表明，在HeLa、Cos-1和F9野生型细胞中，RA存在时会发生转录激活。RARE发生突变后，这种激活以及基础启动子活性均被消除。对P450RAI启动子的缺失和突变分析以及DNase I足迹研究揭示了启动子保守区域中其他几种蛋白质的潜在结合位点。此外，还研究了RARE两侧两个保守的5'-TAAT-3'序列在P450RAI启动子活性中的潜在重要性。此外，这些研究揭示了位于RARE上游的富含G的元件（称为GGRE）对RA诱导性的基本要求。使用来自小鼠F9或P19细胞的核提取物证明该元件可与Sp1和Sp3形成复合物。总之，这些结果表明P450RAI-RARE是非典型的，因为保守的侧翼序列可能在调节RA诱导性和P450RAI（CYP26）的表达中起非常重要的作用。
