ANALYSIS OF THE HUMAN MYELIN BASIC PROTEIN PROMOTER

髓鞘碱性蛋白（myelin basic protein，MBP）是一种丰富的髓鞘结构蛋白， 表达是组织特异性的和发育调节的， 中枢神经系统（CNS）中的少突胶质细胞（OL）。和雪旺 细胞（SC）在周围神经系统（PNS）。 MBP的表达是 对正常中枢神经系统髓鞘形成至关重要。 MBP在这两种组织中的表达 细胞类型在转录起始水平受到调节。 先前对人MBP基因5 '侧翼区的分析表明， 在原代OL和 生长因子扩增的原代SC的培养证明，149 转录起始的bp 5 '含有足以 直接组织特异性，发育调节的MBP表达， 这些细胞。 该提案概述了进一步确定和 表征该区域内的顺式作用元件和反式作用元件， 这些因素将他们联系在一起。 这些实验包括瞬态 正常和改变的MBP启动子-报告基因质粒的转染 在OL、SC和非MBP产生细胞中;以及生化分析 电泳迁移率变动分析，DNA酶I 足迹法，DNA甲基化干扰;并最终筛选 特定反式作用因子的合适表达文库。 这 分析将扩展到MBP中假定的远端增强子 使用相同的技术进行调控。 所确定的要素 最后，将OL的功能与SC中的功能进行比较。 顺式作用元件将在转基因小鼠实验中得到证实 使用MBP启动子-LacZ融合转基因并分析Lac-Z表达 在发育中的CNS和PNS中。 由于髓磷脂基因的调控 在发育过程中的表达可能类似于髓鞘基因的调控 髓鞘再生中的表达，这些研究将确定分子 对理解和改善这些问题可能很重要的机制 脱髓鞘疾病中的髓鞘再生，特别是在CNS中， PNS脱髓鞘

Myelin basic protein (MBP) is an abundant myelin structural protein whose expression is tissue-specific and developmentally-regulated in oligodendrocytes (OL) in the central nervous system (CNS). and Schwann cells (SC) in the peripheral nervous system (PNS). MBP expression is essential for normal CNS myelination. Expression of MBP in both of these cell types is regulated at the level of initiation of transcription. Previous analysis of the 5' flanking region of the human MBP gene in transient transfection studies in developing cultures of primary OL and cultures of growth factor-expanded primary SC have demonstrated that 149 bp 5' of the initiation of transcription contains elements sufficient to direct tissue-specific, developmentally-regulated expression of MBP in these cells. This proposal outlines experiments to further identify and characterize the cis-acting elements within this region and the trans- acting factors which bind them. These experiments include transient transfections of normal and altered MBP promoter-reporter gene plasmids in OL, SC and non-MBP producing cells; as well as biochemical analysis of the MBP promoter by electrophoretic mobility shift assays, DNase I footprinting, DNA methylation interference; and ultimately screening of appropriate expression libraries for specific trans-acting factors. This analysis will be extended to a putative distal enhancer in the MBP regulatory region using the same techniques. The elements identified in OL will be compared to those in SC. Finally, the function of putative cis-acting elements will be confirmed in transgenic mouse experiments using MBP promoter-Lac Z fusion transgenes and analyzing Lac-Z expression in both the developing CNS and PNS. Since the regulation of myelin gene expression in development may be similar to the regulation of myelin gene expression in remyelination, these studies will identify molecular mechanisms potentially important to understanding and improving remyelination in demyelinating diseases, especially in CNS as compared to PNS demyelination.