ANALYSIS OF THE HUMAN MYELIN BASIC PROTEIN PROMOTER

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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.