Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1

BackgroundMitochondria-localized glutamic acid-rich protein (MGARP) is a novel mitochondrial transmembrane protein expressed mainly in steroidogenic tissues and in the visual system. Previous studies showed that MGARP functions in hormone biosynthesis and its expression is modulated by the HPG axis. Methodology/Principal FindingsBy bioinformatics, we identified two characteristic GC-rich motifs that are located proximal to the transcription start site (TSS) of MGARP, and each contains two Specificity protein 1 (Sp1) binding elements. We then determined that the −3 kb proximal MGARP promoter is activated in a Sp1-dependent manner using reporter assays and knockdown of Sp1 led to decreased expression of endogenous MGARP messages. We also demonstrated that one of the two GC-rich motifs, GC-Box1, harbors prominent promoter activity mediated by Sp1, and that it requires both GC boxes for full transcriptional activation. These findings suggest a dominant role for these GC boxes and Sp1 in activating the MGARP promoter through a synergistic mechanism. Consistently, the results of an Electrophoretic Mobility Gel Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) confirmed that Sp1 specifically interacts with the GC-rich region. We further found that estrogen receptor α (ERα), a known Sp1 co-activator, could potentiate GC-boxes containing MGARP promoter activity and this effect is mediated by Sp1. Knockdown of Sp1 significantly diminished the MGARP promoter transactivation and the expression of endogenous MGARP mediated by both Sp1 and ERα. Conclusions/SignificanceThe present study identified a proximal core sequence in the MGARP promoter that is composed of two enriched Sp1 binding motifs and established Sp1 as one major MGARP transactivator whose functions are synergistic with ERα, providing a novel understanding of the mechanisms of MGARP gene transcriptional regulation.背景：线粒体富含谷氨酸蛋白（Mitochondria-localized glutamic acid-rich protein, MGARP）是一种新型线粒体跨膜蛋白，主要表达于类固醇生成组织与视觉系统。既往研究表明，MGARP参与激素生物合成过程，且其表达受下丘脑-垂体-性腺（HPG）轴调控。 研究方法与结果：通过生物信息学分析，我们在MGARP的转录起始位点（transcription start site, TSS）近端区域鉴定出两个特征性GC富集基序，每个基序均包含两个特异性蛋白1（Specificity protein 1, Sp1）结合元件。随后通过报告基因实验证实，MGARP启动子−3 kb近端区域的激活依赖于Sp1；敲低Sp1会导致内源性MGARP信使RNA（mRNA）的表达水平下调。我们还发现，两个GC富集基序中的GC-Box1具有Sp1介导的显著启动子活性，且完整的转录激活需要两个GC盒协同发挥作用。上述结果提示，这些GC盒与Sp1通过协同机制激活MGARP启动子，发挥主导调控作用。与之相符的是，凝胶迁移率变动实验（Electrophoretic Mobility Gel Shift Assay, EMSA）与染色质免疫共沉淀（Chromatin Immunoprecipitation, ChIP）的结果证实，Sp1可特异性结合该GC富集区域。我们进一步发现，作为已知的Sp1共激活因子，雌激素受体α（estrogen receptor α, ERα）可增强携带GC盒的MGARP启动子活性，且该效应由Sp1介导。敲低Sp1可显著削弱Sp1与ERα介导的MGARP启动子反式激活及内源性MGARP的表达。 结论与意义：本研究鉴定出MGARP启动子近端的核心序列，该序列由两个富集Sp1结合位点的基序组成，并证实Sp1是MGARP的主要反式激活因子之一，其功能可与ERα协同发挥作用，为阐明MGARP基因的转录调控机制提供了全新的认知。