INBRE

Myelination involves the coordinate expression of a small set of “myelin-specific” genes. One of these genes, the myelin proteolipid protein gene (Plp), provides an excellent model to study gene regulation for many reasons. First, the Plp gene is abundantly expressed; its gene products constitute roughly 50% of the total protein found in adult CNS myelin. Second, its expression is cell type-specific, being restricted primarily to oligodendrocytes. Third, the gene is regulated during development, with peak expression in rodents occurring during the early postnatal period. Fourth, accurate expression of the gene is essential, with both underexpression and overexpression leading to abnormalities in myelin structure and synthesis and resulting in dysmyelinating or demyelinating disorders in both human diseases and transgenic rodent models. Elucidation of the mechanisms controlling Plp gene expression will be important in the design of effective therapeutic regimens for patients with dysmyelinating or demyelinating diseases. Deletion-transfection analyses in several laboratories have localized positive and negative transcriptional regulatory elements within the Plp promoter and 5’-flanking regions, but none of these elements have been adequate to direct oligodendrocyte-specific expression of the Plp gene. Recent work in the laboratory of Dr. Patricia Wight at the University of Arkansas for Medical Sciences (UAMS) has demonstrated the fundamental importance of regulatory elements/regions, located within the first intron, in the spatial and temporal regulation of Plp gene expression. One of these elements, designated the antisilencer/enhancer (ASE), appears to be crucial for Plp gene expression in oligodendrocytes, particularly with respect to the dramatic surge in expression seen during the active myelination period. Another element appears to be important in mediating repression in cells that do not typically express the gene. Whether this “cell type-specific” negative regulatory element or the ASE are functional in non-oligodendroglial cells that express the Plp gene has yet to be determined. We hypothesize that the spatiotemporal regulation of Plp gene expression in oligodendrocytes is mediated through the ASE. More specifically, we believe that assembly of a higher-order, three-dimensional transcription factor complex (i.e. “enhanceosome”) on the ASE is required to achieve the precise stage-specific regulation observed in myelinating oligodendrocytes. In addition, we hypothesize that other elements within the first intron and/or 5’ flanking sequences are more important than the ASE in regulating Plp gene expression in non-oligodendroglial cell types. To test our hypotheses, we will characterize the effects of Plp intron 1 DNA on Plp gene expression in rat CG-4 cells, which can be induced to differentiate into oligodendrocytes, and in mouse TM3 cells, a non-oligodendroglial cell line that expresses the gene. Secondly, we will examine the chromatin structure of the ASE within endogenous Plp intron 1 DNA in three Plp expressing cell lines [N20.1, CG-4, and TM3] and in a non-expressing liver cell line (+/+ Li). Undergraduate students, under the supervision of Dr. Brian Greuel at John Brown University, will be primarily responsible for addressing the first of these two specific aims. They will transiently transfect the TM3 cells with an existing series of Plp-lacZ constructs that have variable lengths of Plp intron 1 sequences, combined with Plp 5’ flanking sequences, driving expression of the lacZ reporter gene. CG-4 cells will be stably transfected in their progenitor stage with a similar series of Plp-lacZ constructs and then analyzed in both their progenitor and differentiated stages. Relative expression of the reporter gene will be assessed by measuring b-galactosidase activity in cell extracts of transfected cells. The second specific aim will be the focus of Dr. Greuel in work performed this summer in Dr. Wight’s laboratory at the UAMS and during the following academic year at John Brown University. Again, rat CG-4 cells will be tested in both the progenitor and fully differentiated stages. Analysis of chromatin structure will be accomplished by in vivo footprinting, in which single-stranded cuts are introduced in regions of DNA that are unprotected by the binding of transcription factors in vivo. The sites of cutting will then be mapped by ligation-mediated and terminal deoxynucleotidyl transferase-dependent polymerase chain reactions (LMPCR and TDPCR), combined with DNA sequencing. These studies will be important in identifying changes in chromatin structure that are associated with the spatiotemporal regulation of Plp gene expression.

Back

Updated 10/31/2005

The Arkansas INBRE is Supported by a grant from the National Institutes of Health
and the National Center for Research Resources (P20 RR-16460).

Please contact Caroline Miller Robinson regarding questions or comments about this site or our program. For more information about the University of Arkansas for Medical Sciences visit http://www.uams.edu.