Single-stranded DNA-binding complex involved in transcriptional regulation of mouse μ-opioid receptor gene
Single-stranded DNA-binding complex involved in transcriptional regulation of mouse μ-opioid receptor gene
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DOI：
10.1074/jbc.m004279200
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发表时间：
2001-01-05
影响因子：
4.8
通讯作者：
Loh, HH
中科院分区：
文献类型：
--
作者：
Ko, JL;Loh, HH
在此之前，我们报道了小鼠μ阿片受体（莫尔）基因中存在双（远端和近端）启动子，小鼠脑中的mar转录主要由近端启动子启动。Sp因子与双链顺式调控元件结合，对近端启动子活性至关重要。在这里，我们进一步报告，一个单链（SS）顺式调节元件和反式作用蛋白因子也是重要的近端启动子活性。一个26-bp的mar聚嘧啶/聚嘌呤区（PPy/u）可以采用ss DNA构象，如S1核酸酶敏感性所示。使用电泳迁移率变动分析与核提取物从MOR表达SH-SY 5 Y细胞，我们表明，有义链的PPy/u相互作用的主要核蛋白，称为莫尔聚嘧啶结合蛋白（MPY），这是不相关的SP因子。Southwestern印迹分析表明，mPy蛋白的大小与25 kDa相似。功能分析表明，mPy蛋白可以反式激活mar启动子以及异源启动子。此外，ss（mPy）和ds（Sps）DNA结合因子的组合激活，与重叠的DNA（PPy/u）区域相互作用，是近端启动子激活所必需的。因此，我们的研究结果表明，小鼠mar基因的转录调控的se和ds DNA结合因子的相互作用。
Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu -opioid receptor (mor) gene, with mar transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mar polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is similar to 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mar promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mar gene is regulated by an interplay of se and ds DNA binding factors.