Transcriptional regulation of mouse μ opioid receptor gene:: Sp3 isoforms (M1, M2) function as repressors in neuronal cells to regulate the μ opioid receptor gene
Transcriptional regulation of mouse μ opioid receptor gene:: Sp3 isoforms (M1, M2) function as repressors in neuronal cells to regulate the μ opioid receptor gene
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DOI：
10.1124/mol.104.008284
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发表时间：
2005-05-01
影响因子：
3.6
通讯作者：
Loh, HH
中科院分区：
文献类型：
--
作者：
Choi, HS;Hwang, CK;Loh, HH
小鼠MU阿片受体(MOR)基因的5‘侧翼区有两个启动子，分别称为远端和近端。MOR基因主要由近端启动子启动。此前，一些重要的顺式元件和反式因子已被证明在MOR基因的近端启动子中发挥功能作用。在这项研究中，我们定义了位于启动子近端-219-189碱基对(翻译起始点设计为+1)区域的另一个功能性负调控元件。它被指定为Sp结合序列，因为其序列与共同的Sp结合元件同源性。Sp结合元件的突变导致MOR阳性细胞(NMB细胞)中MOR启动子活性100%增加，证实了Sp结合序列的负作用。令人惊讶的是，电泳迁移率改变分析和染色质免疫沉淀分析表明，SP3及其异构体(M1和M2)与Sp结合序列具有特异性结合。在用编码Sp1、SP3和SP3的M1和M2亚型的cDNA共转染果蝇SL2细胞的实验中，M1和M2亚型反式抑制MOR启动子，而Sp1和SP3反式激活MOR启动子。值得注意的是，异位表达SP3的M1和M2亚型导致内源性MOR基因转录本在NMB细胞中受到抑制。这些结果表明，SP3转录因子的M1和M2亚型与Sp结合序列的结合可能在小鼠MOR基因的表达中起作用。
The 5'-flanking region of the mouse mu opioid receptor (MOR) gene has two promoters, referred to as distal and proximal. MOR mRNA is predominantly initiated by the proximal promoter. Previously, several important cis-elements and transfactors have been shown to play a functional role in the proximal promoter of the MOR gene. In this study, we defined another functional, negative regulatory element located in the -219- to -189- base pair (translational start site designed as +1) region of the proximal promoter. It is designated as the Sp binding sequence for its sequence homology to the consensus Sp binding element. Mutation of the Sp binding element led to a 100% increase of MOR promoter activity in MOR-positive cells (NMB cells), confirming the negative role of the Sp binding sequence. Surprisingly, electrophoretic mobility shift analysis and chromatin immunoprecipitation assays revealed that Sp3 and its isoforms (M1 and M2) were specifically bound to the Sp binding sequence. In cotransfection assays of Drosophila melanogaster SL2 cells using cDNA encoding Sp1, Sp3, and the M1 and M2 isoforms of Sp3, the M1 and M2 isoforms trans-repressed the MOR promoter, whereas Sp1 and Sp3 trans-activated the MOR promoter. Significantly, ectopic expression of the M1 and M2 isoforms of Sp3 led to repression of the endogenous MOR gene transcripts in NMB cells. These results suggest that the binding of the M1 and M2 isoforms of the Sp3 transcription factor to the Sp binding sequence may play a role in mouse MOR gene expression.