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Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the proximal E1b far upstream promoter

机译:Sp1和Sp3转录因子通过与远端启动子E1b的相互作用来调节人微粒体环氧水解酶(EPHX1)的基础表达

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摘要

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The proximal E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide −300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.
机译:微粒体环氧水解酶(mEH,EPHX1)是一种关键的生物转化酶,催化许多异种生物的代谢。使用替代启动子转录人类mEH。近端E1启动子在肝脏中有活性,而远端E1b启动子驱动mEH在包括肝脏在内的所有组织中的表达。尽管已经在E1转录的调控中发现了几种肝脏特异性转录因子,但关于E1b转录调控的机制知之甚少。 DNase I超敏位点的全基因组定位揭示了E1b上游-300核苷酸与下游+400核苷酸之间的开放染色质区域。该区域与先前描述的负责维持高基础启动子活性的启动子区域一致。在对该位置的计算机分析中发现了几个Sp1 / Sp3结合位点。这些基序的定点诱变抑制了E1b近端启动子的反式激活活性,表明它们作为E1b启动子调控的重要因素。此外,Sp1和Sp3过表达后,E1b启动子活性显着增加,而选择性Sp1抑制剂光神霉素A降低了启动子活性。 EMSA研究表明,Sp1与两个假定的Sp1 / Sp3结合位点结合。 ChIP分析证实,内源性Sp1和Sp3均与E1b的近端启动子区域结合。使用siRNA敲除Sp1表达不会改变内源性E1b转录水平,而敲除Sp3则大大降低了不同人细胞系中E1b的表达。综上所述,这些结果支持了这样的概念,即Sp1和Sp3在功能上参与了调节衍生mEH E1b变异转录本的基础表达的转录整合子。

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