Peng Hui, Zhu Qin-Shi, Zhong Shuping, Levy Daniel
University of Southern California, Keck School of Medicine, Department of Biochemistry and Molecular Biology, 2011 Zonal Avenue, Los Angeles, CA 90033, USA.
Biochim Biophys Acta. 2013 Oct;1829(10):1000-9. doi: 10.1016/j.bbagrm.2013.05.003. Epub 2013 May 26.
Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα-NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.
微粒体环氧化物水解酶（mEH）是一种双功能蛋白，在多种异源生物的代谢中起核心作用，同时介导胆汁酸依赖钠的转运进入肝细胞，在肝细胞中胆汁酸参与胆固醇排泄和代谢、脂质消化以及调节众多信号通路。先前的研究已经证明GATA-4和C/EBPα-NF/Y复合物在mEH基因（EPHX1）的调控中起关键作用。在本研究中，我们发现肝细胞核因子4α（HNF-4α）以及组成型雄烷受体/视黄酸X受体（CAR/RXR）也结合至近端启动子区域并调节EPHX1的表达。抑制HNF-4α表达的胆汁酸也会降低EPHX1的表达。研究还证实，HNF-4α的结合对于CAR激活EPHX1活性至关重要，这表明这些相邻因子之间形成了复合物。使用该区域的生物素化寡核苷酸结合生物磁珠和质谱分析进一步探索了这种调节复合物的性质，结果表明存在一种额外的抑制因子（多聚嘧啶结合蛋白相关剪接因子，PSF），通过免疫共沉淀和染色质免疫沉淀分析得到证实，该抑制因子与结合DNA的CAR/RXR/HNF-4α相互作用，形成一种四组分调节复合物。
