Regulation of MeCP2 by CREB-induced microRNAs

描述(申请人提供)：Rett综合征是一种神经发育障碍，典型的原因是甲基CpG结合蛋白MeCP2的突变。MeCP2生物学的一个相对较少受到关注的方面是它在翻译水平上的调节。MeCP2转录本的长异构体(大脑中发现的主要转录本)包含高度保守的进化序列块，包括一些数千万年来没有发生过单核苷酸变化的区域。我们认为这些序列是一个microRNAs家族的结合位点，其表达由关键的信号依赖转录激活因子CREB驱动。我们的总体假设是，CREB指导一系列调控MeCP2翻译的microRNAs的表达。CREB刺激microRNA表达的能力反过来又受到REST的控制，REST是众所周知的神经基因抑制因子。该模型为CREB(以及激活CREB功能的途径)提供了一种长期调节MeCP2蛋白水平的机制。这一机制被认为是为了防止在某些类型的神经元活动后可能发生的MeCP2蛋白水平升高的有害影响。 我们的具体目标是：1)确定5个CREB调控的microRNAs，即miR132、miR191、miR320、miR219-1和miR328是否抑制MeCP2蛋白的表达。特定的2‘-O-甲基寡核苷酸将单独和联合使用，以确定它们是否阻止microRNA介导的MeCP2水平的变化。还将确定microRNAs对MeCP2与BDNF启动子结合的影响；2)表征调节五个microRNAs表达的发育和信号通路；3)确定miR132对MeCP2的发育调控是否受REST控制。病毒学上对microRNAs的转录调控或控制MeCP2蛋白表达的因素一无所知。这项建议中描述的研究解决了一种全新的基因调控机制，这对理解MeCP2生物学和一般的神经发育具有重要意义。

DESCRIPTION (provided by applicant): Rett syndrome is a neurodevelopmental disorder typically due to mutations in the methyl CpG binding protein, MeCP2. An aspect of MeCP2 biology that has received relatively little attention is its regulation at the level of translation. The long isoform of the MeCP2 transcript (the predominant transcript found in brain) contains blocks of sequence that are highly conserved evolutionary, including some regions that have not undergone a single nucleotide change over tens of millions of years. We propose that these sequences are binding sites for a family of microRNAs whose expression is driven by CREB, a critical signal-dependent transcriptional activator. Our overall hypothesis is that CREB directs the expression of a family of microRNAs that regulate MeCP2 translation. The ability of CREB to stimulate microRNA expression is, in turn, under the control of REST, a well-known represser of neural genes. This model provides a mechanism for CREB (as well as the pathways that activate CREB function) to regulate MeCP2 protein levels over the long term. This mechanism is proposed to prevent the deleterious effects of increased MeCP2 protein levels that may occur after certain types of neuronal activity. Our specific aims are to 1) Determine whether five CREB-regulated microRNAs, namely miR132, miR191, miR320, miR219-1, and miR328 inhibit MeCP2 protein expression. Specific 2'-O-methyl oligonucleotides will be used singly and in combination to determine whether they block the microRNA-mediated changes in MeCP2 levels. The effects of the microRNAs on the binding of MeCP2 to the BDNF promoter will also be determined;-2) Characterize the developmental and signaling pathways that regulate expression of the five microRNAs; 3) Determine whether the developmental regulation of MeCP2 by miR132 is controlled by REST. There is virutally nothing known about the transcriptional regulation of microRNAs or the factors that control MeCP2 protein expression. The studies described in this proposal address a completely novel mechanism of gene regulation that has implications for the understanding of MeCP2 biology and neural development in general.