中枢神经系统髓鞘形成过程中基于Nkx2.2的转录及转录后调控研究

少突胶质细胞（OLs）形成的髓鞘能确保神经冲动在神经元中快速精准地传导，髓鞘形成过程受转录及转录后水平的严格调控。Nkx2.2和QKI分别是该过程的一个重要转录因子和具有转录后调控功能的RNA结合蛋白，本项目在获知QKI很可能是Nkx2.2调控的靶基因的基础上，通过在特定时期诱导Nkx2.2的过表达和缺失，明确Nkx2.2对QKI的表达及其转录后调控的影响；通过QKI启动子组蛋白乙酰化水平检测，Nkx2.2与QKI启动子结合的ChIP-PCR分析，以及两者的结合是否是HDAC依赖性的体外细胞实验，确定在OLs形成髓鞘过程中，Nkx2.2调控QKI的分子机制；利用IP结合RT-PCR和EMSA在体外细胞中检测QKI能否结合Nkx2.2的mRNA而进行转录后调控。以上研究有助于我们理解转录和转录后调控协同作用在调控OLs髓鞘化中的重要性，为提出髓鞘相关疾病的治疗新方法提供理论基础和分子手段。

Oligodendrocyte cells (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials among neurons. The lineage progression from oligodendrocyte progenitor cells (OPCs) to mature myelinating OLs was controled both by transcriptional and post-transcriptional regulation. Nkx2.2 is the critical transcription regulator that controls the timing of OLs differentiation and myelination. In addition, the sophisticated posttranscriptional regulation by QKI is essential for OL and myelin development. In this grant, we will investigate the effect of overexpression/inactivation of Nkx2.2 on the expression of QKI and its post-transcriptional regulation in oligodendrocyte myelination based on preliminary results that QKI is likely to be the downstream target gene of Nkx2.2 in the process of myelination. At the same time we will examine whether knockdown or overexpression of Nkx2.2 in the OLs may affect HDAC-mediated histone acetylation at the QKI promoter as a co-operating mechanism in regulating QKI transcription in vivo. In addition, we will perform ChIP-PCR and Luciferase reporter experiments in the OLs cell line to directly test the effect of Nkx2.2 on QKI promoter activity. Futhermore, overexpression/RNA-interference of HDACs in OLs line following ChIP-PCR will be used to test whether Nkx2.2 represses QKI transcription/expression in an HDACs dependent manner. On the other hand, we will express exogenous QKI in the OLs cell line, via the IP associate with RT-PCR and EMSA to directly access whether QKI can bind to Nkx2.2. Then, qRT-PCR and immunoblot will be carried out to quantify Nkx2.2 mRNA and protein expression levels to test the post-transcriptional regulation of QKI on Nkx2.2. This study will help us better understand the importance of the functional interplay between transcriptional and post-transcriptional regulation in myelinogenesis, and provide important theoretical guidance and molecular strategies for the treatment of clinical myelin associated diseases.