State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.
PLoS One. 2012;7(11):e50053. doi: 10.1371/journal.pone.0050053. Epub 2012 Nov 27.
Mitochondria-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 FINDINGS: By 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/SIGNIFICANCE: The 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.
线粒体定位的谷氨酸丰富蛋白（MGARP）是一种新型的线粒体跨膜蛋白，主要在类固醇生成组织和视觉系统中表达。先前的研究表明，MGARP 参与激素生物合成，其表达受 HPG 轴调节。
方法/主要发现：通过生物信息学，我们鉴定了两个位于 MGARP 转录起始位点（TSS）近端的特征性富含 GC 基序，每个基序包含两个特异性蛋白 1（Sp1）结合元件。然后，我们通过报告基因检测和 Sp1 敲低确定，-3kb 近端的 MGARP 启动子以 Sp1 依赖的方式被激活，Sp1 敲低导致内源性 MGARP 消息的表达减少。我们还证明，两个富含 GC 基序之一的 GC-Box1 具有由 Sp1 介导的显著启动子活性，并且它需要两个 GC 盒才能实现完全转录激活。这些发现表明这些 GC 盒和 Sp1 在通过协同机制激活 MGARP 启动子方面发挥主导作用。一致地，电泳凝胶阻滞分析（EMSA）和染色质免疫沉淀（ChIP）的结果证实 Sp1 特异性与富含 GC 的区域相互作用。我们进一步发现，雌激素受体 α（ERα），一种已知的 Sp1 共激活因子，能够增强含有 MGARP 启动子活性的 GC 盒，这种作用是通过 Sp1 介导的。Sp1 敲低显著减弱了 Sp1 和 ERα 介导的 MGARP 启动子的转录激活和内源性 MGARP 的表达。
结论/意义：本研究鉴定了 MGARP 启动子中一个近端核心序列，该序列由两个富含 Sp1 结合基序组成，并确定 Sp1 是 MGARP 的主要转录激活因子之一，其功能与 ERα 协同，为 MGARP 基因转录调控的机制提供了新的认识。
