Abstract Dr. B. Greuel
Title of Project Transcriptional Regulation of the Myelin Proteolipid Protein Gene: Effect of Intron 1-Binding Factors on Chromatin Remodeling During Oligodendrocyte Differentiation
Abstract Expression of the myelin proteolipid protein (Plp) gene in mammals is intimately tied to the differentiation of oligodendrocytes, the myelin-producing cells of the central nervous system. Overexpression or mutation of the Plp gene leads to oligodendrocyte death and can be the cause of both dysmyelinating and demyelinating disorders in humans and rodents. Understanding the molecular mechanisms involved in Plp gene regulation may provide insights into the process of oligodendrocyte differentiation and suggest possible therapeutic approaches for the human neurological disorders. The first specific aim of this project is to identify short sequence motifs that are essential for the function of a key regulatory element, the antisilencer/enhancer (ASE), found within the first intron of the Plp gene. A series of 8 bp linker-scan mutations created within the ASE region will be analyzed by electrophoretic mobility shift assays for their effects on nuclear factor binding. Effects on transcriptional activity will be measured in transient expression assays using lacZ as a reporter gene in cultured oligodendroglial cells. The second specific aim is to examine the chromatin structure of the ASE region in non-expressing cells and in oligodendrocytes at different stages of cell differentiation. In this approach, intact cells will be treated with dimethylsulfate (DMS) and fragments of the modified DNA will be amplified by ligation-mediated PCR and analyzed by DNA sequencing. The third specific aim is to analyze the effects of upregulating or downregulating the activities of candidate ASE-binding factors on Plp gene expression and chromatin structure. To assess the effects of upregulating transcription factor activity, oligodendroglial cells will be transiently cotransfected with Plp-lacZ reporter gene constructs and expression vectors designed to overexpress specific transcription factors. An RNA interference (RNAi) approach will be used to lower the expression of these factors. The impact of these manipulations on chromatin structure at the endogenous Plp locus in cultured oligodendroglial cells will then be examined by in vivo footprinting.
Updated 07/31/2006
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