GENETIC ELEMENTS REGULATIONG MYELIN GENE EXPRESSION

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Recent studies in our laboratory have shown that the 32 kb myelin basic protein (MBP) transcription unit is contained within an overlapping transcription unit, termed the Golli-mbp gene, which is approximately 105 kb in the mouse. The Golli-mbp transcription unit consists of eleven exons of which parts or all of the last seven exons form the MBP transcription unit. Expression of the MBP transcription unit begins at a promoter upstream of exon 5b of the Golli-mbp gene, and expression of the Golli-mbp transcription unit begins at exon 1, producing a number of tissue, cell and developmentally regulated splice products at least three of which contain one or more exons found within the MBP transcription unit. The overall objective of this proposal is to determine the developmental and tissue-specific regulatory element of the Golli-mbp transcription unit in the mouse using in vitro transfection of cells and transgenic mice. We will analyze the role of these elements in modulating the expression of the MBP transcription with respect to levels of transcription as well as the tissue, cell and developmental expression of the gene. Initially, we will analyze the region upstream of Golli-mbp exon 1 for elements involved in the tissue, cell and developmental regulation of this transcription unit. We w ill also examine the possibility that one or more of these regulatory elements are found elsewhere in the gene, particularly the first intron. We will prepare constructs of putative regulatory regions plus a reporter gene and test them by transfection analysis in cell lines that do and do not express the Golli-mbp gene. We will then test the most promising constructs in transgenic animals to provide proof that the putative regulatory regions are operative in vivo. In other experiments we will examine the influence of the Golli-mbp promoter and any other regulatory elements we find on the levels and nature of expression of the MBP transcription unit. The deletion in the shiverer mouse genome affects at least two products of the Golli-mbp transcription unit in addition to MBPs. In view of the pleiotropic nature of the shiverer deletion on oligodendrocyte function we will introduce a Golli-mbp minigene into shiverer mice and carefully examine all the effects known to be associated with the shiverer mutation (beyond MBP expression and the "shivering" phenotype). These studies could help us define the consequences of the expression of the Golli-mbp gene products. They would also help us determine if the produces themselves had an effect on the expression of the MBP transcription unit during oligodendrocyte development.