The myelin proteolipid protein gene (Plp1) encodes the most abundant protein present in mature myelin from the central nervous system (CNS). Its expression in oligodendrocytes is temporally regulated, dramatically peaking during the active myelination period of CNS development. Previous studies with transgenic mice containing Plp1-lacZ fusion genes have demonstrated the importance of Plp1 intron 1 DNA in mediating temporal regulation of transgene expression in brain, in a manner consistent to that of the endogenous Plp1 gene. Deletion-transfection analysis with Plp1-lacZ constructs resulted in the identification of a single positive regulatory element within Plp1 intron 1, which is operational in N20.1 cells (an immortalized mouse oligodendroglial cell line). We named this element ASE for antisilencer/enhancer based on its properties in N20.1 cells. The primary focus of this dissertation project was to understand the role of the ASE in regulating Plp1 gene expression in oligodendrocytes during development.
Initially, the N20.1 and Oli-neu cell lines were characterized as models of oligodendroglial cells in which to study the regulation of myelin-related genes. Comparisons between the two cell lines place them at slightly different stages of oligodendrocyte maturation. The mRNA splice isoforms generated from the examined myelin-related genes, along with the relative amounts of protein expressed by these genes, suggests that while both cell lines are representative of immature oligodendrocytes, Oli-neu cells appear to be further along the differentiation pathway. Transfection analysis with Plp1-lacZ constructs that either contain the ASE, or are missing it, demonstrated that the ASE is functional in both N20.1 and Oli-neu cells and seems to have a more potent effect in Oli-neu cells. Although the ASE appears to be the sole positive regulatory element within Plp1 intron 1 DNA that functions in N20.1 cells, the intron also contains an additional positive regulatory element (or elements) that are active in Oli-neu cells.
Next, the ASE was deleted from the native Plp1 gene in mouse using a Cre/lox strategy in order to study the role of the ASE in vivo. Although the ASE was critical to obtain high levels of Plp1-lacZ expression in transfected N20.1 and Oli-neu cells, deletion of the ASE from the native Plp1 gene in mouse had only a minor effect on the developmental profile of Plp1 gene expression in brain. Results presented in this dissertation suggest that the ASE is functional in vivo, but other regulatory element(s) exist, which are capable of mediating correct temporal expression, even in the absence of the ASE from the Plp1 gene. Perhaps there is functional redundancy between different regulatory elements which can compensate for the lack of the ASE. Alternatively, the influence of the ASE may be much stronger in (Plp1-lacZ) transfected cells, when compared to its effect on the native gene in vivo. Furthermore, deletion of the ASE from the native gene had only a minimal effect on Plp1 gene expression, in brain, during the remyelination phase of cuprizone-induced demyelination.
Finally the role for Sox10, a critical gliogenic factor, was examined in the regulation of the Plp1 gene expression. Transient transfection with a plasmid expressing a dominant-negative form of Sox10 markedly reduced endogenous Plp1 gene expression in Oli-neu cells. Furthermore, co-transfection with various Sox10 mutants and Plp1-lacZ constructs (containing either all, some, or none of Plp1 intron 1 DNA) resulted in decreased levels of β-galactosidase activity in a manner consistent with their clinical manifestations in humans. Results presented here demonstrate that the Sox10 mutants may act through regulatory elements located within Plp1 intronl DNA.
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 71/10, Dissertation Abstracts International|
|Subjects:||Molecular biology, Neurosciences, Physiology|
|Keywords:||Antisilencer/enhancer, Demyelination, Gene targeting, Myelin proteolipid protein, Oligodendrocytes|
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