Identification of sterol-independent regulatory elements in the human ATP-binding cassette transporter A1 promoter -: Role of Sp1/3, E-box binding factors, and an oncostatin M-responsive element
Identification of sterol-independent regulatory elements in the human ATP-binding cassette transporter A1 promoter -: Role of Sp1/3, E-box binding factors, and an oncostatin M-responsive element
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DOI：
10.1074/jbc.m110270200
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发表时间：
2002-04-26
影响因子：
4.8
通讯作者：
Schmitz, G
中科院分区：
文献类型：
--
作者：
Langmann, T;Porsch-Özcürümez, M;Schmitz, G
ATP结合盒转运体A1(ABCA1)在人单核细胞中表现出分化、cAMP和类固醇依赖性的上调。作为正在进行的研究的一部分，我们调查了人类和小鼠ABCA1基因之间高度保守的近端启动子区域。利用报告基因分析，我们发现转录起始点上游的TATA盒24对于RAW 264.7和HepG2细胞中的启动子活性是必不可少的，而转录活性的进一步增强是由启动子区域-175介导的。凝胶位移分析显示Sp1与a-91GNC基序结合，Sp1和SpA与a-157GNC启动子区域结合。在用果蝇S2细胞进行的共转染实验中，我们证明了SpA与Sp1竞争结合-157GNC基序并发挥阻遏作用。另一方面，Sp1的过表达增加了HeLa细胞ABCA1mRNA的表达，并增加了RAW 246.7巨噬细胞的胆固醇和磷脂外流。我们还表明，保守的E-box结合上游刺激因子1和2以及肝脏核因子1a，并且E-box的突变增强了RAW 264.7细胞的结构性ABCA1的表达，这意味着该元件在沉默ABCA1的表达中发挥了作用。除了对基础基因表达的功能重要性外，我们还发现核心启动子区域(-175到+224)也是细胞因子癌基因M诱导ABCA1表达的原因，从而导致HepG2细胞ABCA1mRNA水平的快速上升。有趣的是，这种由抑癌素M诱导的表达并不依赖于目前已知的ABCA1启动子中的序列基序。综上所述，在人类ABCA1启动子近端与转录因子Sp1/3、上游刺激因子1和2以及肝脏核因子1a相关的顺式元件的功能复合体已经被鉴定，这使得对ABCA1基因表达的微妙的组织特异性调控成为可能。
The ATP-binding cassette transporter A1 (ABCA1) shows a differentiation-, cAMP-, and sterol-dependent up-regulation in human monocytes. As part of an ongoing study, we investigated the proximal promoter regions that are highly conserved between the human and murine ABCA1 genes. Using reporter gene assays, we show here that a TATA box 24 by upstream of the transcription initiation site is essential for promoter activity in RAW 264.7 and HepG2 cells, whereas further enhancement of transcriptional activity is mediated by the -175 by promoter region. Gel shift assays revealed in vitro binding of Sp1 to a -91 GnC motif as well as binding of Sp1 and Spa to a -157 GnC promoter region. In co-transfection experiments using Drosophila S2 cells, we demonstrate that Spa competes with Sp1 for binding to the -157 GnC motif and acts as a repressor. On the other hand, overexpression of Sp1 increased ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. We also show here that the conserved E-box at position -140 binds upstream stimulatory factors 1 and 2 and hepatic nuclear factor la and that mutagenesis of the E-box enhanced constitutive ABCA1 expression in RAW 264.7 cells, implying a role for this element in silencing ABCA1 expression. Besides the functional importance for basal gene expression, we have identified that the core promoter region (-175 to +224) is also responsible for the induction of ABCA1 by the cytokine oncostatin M, resulting in a rapid increase in ABCA1 mRNA levels in HepG2 cells. Interestingly, this oncostatin M-induced expression is not dependent on the currently known sequence motifs in the ABCA1 promoter. In conclusion, a functional complex of cis-elements within the proximal human ABCA1 promoter associated with the transcription factors Sp1/3, upstream stimulatory factors 1 and 2, and hepatic nuclear factor la has been characterized, which allows a subtle tissue-specific regulation of ABCA1 gene expression.