Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.
Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.
复制标题
少突胶质细胞原代培养物中人 MBP 启动子的分析：近端 MBP 启动子中的正向和负向顺式作用元件介导少突胶质细胞特异性的 MBP 表达。
DOI：
10.1002/jnr.490360412
复制
发表时间：
1993
影响因子：
4.2
通讯作者：
Kamholz,J
中科院分区：
文献类型：
--
作者：
Wrabetz,L;Shumas,S;Grinspan,J;Feltri,ML;Bozyczko,D;McMorris,FA;Pleasure,D;Kamholz,J
由于髓鞘碱性蛋白表达的调控主要依赖于转录的启动，因此我们分析了人髓鞘碱性蛋白基因5‘侧翼区在发育中的少突胶质细胞原代培养中的瞬时转染研究。我们证明了转录起始的149个5‘碱基对足以指导少突胶质细胞特异性表达髓鞘碱性蛋白。融合转录本与内源性髓鞘碱性蛋白转录本的编码序列一致，氯霉素乙酰转移酶报告基因在这些细胞中的表达仅限于少突胶质细胞。在这149个碱基对区域中，鉴定出一个远端的负作用片段和一个近端的正作用片段，该片段包含一个共同的核因子I位点。远端片段在Cos-7细胞中的表达比在少突胶质细胞中表现得更负面，将表达水平降低到背景水平。此外，这些功能上重要的顺式作用片段以不同于包括Cos-7细胞在内的其他细胞的模式结合少突胶质细胞核蛋白。有趣的是，远端片段增加了少突胶质细胞中异源SV40启动子的活性，但对Cos-7细胞中的SV40启动子没有影响。我们得出结论，功能阴性的远端片段可能通过限制MBP在其他细胞中的表达来介导MBP在少突胶质细胞中的特异性表达。这些实验强烈支持使用少突胶质细胞的原代培养来分析髓鞘特异性启动子。©1993 Wiley-Liss，Inc.
Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5′ flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5′ of the initiation of transcription was sufficient to direct oligodendrocyte‐specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negativecis‐acting segment, containing a consensus nuclear factor I site, and one proximal, positivecis‐acting segment were identified. The distal segment behaved more negatively in Cos‐7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally importantcis‐acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos‐7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos‐7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte‐specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin‐specific promoters. © 1993 Wiley‐Liss, Inc.