Molecular Basis of mu-Opioid Receptor Gene Regulation

描述（由申请人提供）：这是一份新研究者的修订R01提案（1 R01 DA016673-01）。多阿片受体（MOR）在镇痛中的关键作用，以及在耐受性和依赖性的发展中是公认的。然而，关于铁道部的监管，还有很多需要了解的地方。本研究的目的是研究细胞特异性MOR基因表达的机制，特别是单链DNA结合蛋白及其与双链DNA结合蛋白的功能相互作用在MOR基因调控中的作用。我们之前已经确定了远端和近端MOR启动子，后者优先指导大脑中的MOR表达。在近端启动子区域已鉴定出若干顺式元件，包括ds和ass顺式元件。还显示了Sp （ds结合因子）和一种未识别的ss DNA结合蛋白的参与，该蛋白特异性地结合MOR ss顺元件。这些数据为MOR基因如何在转录水平上调控提供了线索。最近，我们利用酵母单杂交筛选系统，成功地从小鼠脑cDNA文库中克隆出了ass DNA结合蛋白poly - C binding protein （PCBP）。我们的初步研究强烈表明，该克隆的PCBP能够高亲和力地特异性结合MOR的ss顺式元件，并可能参与MOR基因的调控。PCBP从未被证明是一种转录因子，其靶基因也未被确定。PCBP作为转录调节剂的新作用及其与其他转录因子在MOR基因表达调控中的相互作用将在本提案中进行研究。首先将研究PCBP在调节MOR基因表达中的体内功能作用（Specific Aim 1）。PCBP如何在MOR基因调控中作为一种新的转录调节剂的分子基础将在Specific Aim 2中进行研究。PCBP和Sp转录因子在MOR基因调控中的相互作用，以及Sp蛋白和PCBP翻译后修饰对MOR基因调控的影响，将在Specific Aim 3中进行研究。最后，在Specific Aim 4中，我们将研究PCBP是否直接与转录机制（包括RNA聚合酶II和辅助转录因子，如TFIID）相互作用以调节MOR基因表达。从这些研究中获得的信息将为MOR基因调控的分子基础提供新的见解。了解调节MOR基因表达的机制可能有助于开发改变受体表达的方法，并可能进一步帮助开发理解疼痛和药物滥用的新方法。

DESCRIPTION (provided by applicant): This is a revised R01 proposal (1 R01 DA016673-01) of a new investigator. The critical roles of the mu-opioid receptor (MOR) in analgesia, as well as in the development of tolerance and dependence are well-established. However, much remains to be learned about MOR regulation. The goal of this new R01 proposal is to investigate the mechanisms underlying cell-specific MOR gene expression, especially focusing on the roles of single-stranded (ss) DNA binding protein and its functional interaction with double-stranded (ds) DNA binding proteins in MOR gene regulation. We have previously identified a distal and a proximal MOR promoter, with the latter preferentially directing MOR expression in the brain. Several cis-elements have been identified, including the ds and ass cis-elements, in the proximal promoter region. The involvement of both Sp (ds binding factors) and an unidentified ss DNA binding protein, which specifically binds to the MOR ss ciselement, has also been shown. These data provided clues as to how the MOR gene is regulated at the transcriptional level. Recently, we have successfully cloned ass DNA binding protein, poly C binding protein (PCBP), from a mouse brain cDNA library using the yeast one-hybrid screening system. Our preliminary studies strongly suggested that this cloned PCBP can specifically bind to the MOR ss cis-element with high affinity, and may participate in the MOR gene regulation. PCBP has never been documented as a transcription factor, nor has its target gene been identified. The novel role of PCBP as a transcription regulator as well as its interaction with other transcription factors in the regulation of MOR gene expression will be examined in this proposal. The in vivo functional roles of PCBP in the regulation of MOR gene expression will first be examined (Specific Aim 1). The molecular basis of how PCBP can serve as a novel transcriptional regulator in MOR gene regulation will be examined in Specific Aim 2. The interplay (interaction) between PCBP and Sp transcription factors in regulation of MOR gene, as well as the effects of post-translational modifications of Sp proteins and PCBP on MOR gene regulation, will be examined in Specific Aim 3. Finally, in Specific Aim 4, we will examine whether PCBP directly interacts with the transcriptional machinery (including RNA polymerase II and auxiliary transcription factors, such as TFIID) in regulating MOR gene expression. The information gained from these studies will provide new insights into the molecular basis of MOR gene regulation. The understanding of mechanisms regulating MOR gene expression may help in the development of approaches to alter the receptor expression and may further help in the development of novel approaches understanding pain as well as drug abuse.