Role of an intronic enhancer in the regulation of myelin proteolipid protein gene expression

The myelin proteolipid protein (Plp) gene encodes the most abundant protein present in mature myelin from the central nervous system (CNS). Expression of the gene is restricted primarily to oligodendrocytes, with peak expression occurring during the active myelination period of CNS development. Previous studies with Plp-lacZ fusion genes in transgenic mice suggest that regulatory element(s) located within Plp intron 1 DNA are critical for its temporal regulation in the brain. Deletion-transfection analyses conducted in the N20.1 oligodendroglial cell line have identified a single positive regulatory element located in Plp intron 1, dubbed ASE for anti silencer/enhancer. The major goal of this dissertation is to understand mechanistically how the ASE controls Plp gene expression. Although previous studies have demonstrated that the ASE possesses enhancer-like characteristics (orientation-independent; multiple copies of the ASE promote higher levels of Plp-lacZ gene activity), the current studies were designed to test: (1) if the ASE can activate a heterologous promoter; (2) if the ASE is still active when displaced from its normal context (intronic DNA); (3) if orthologous sequences from other species can functionally substitute for the mouse ASE; and (4) if deletion of the ASE from the native gene in mice by targeted mutagenesis affects Plp gene expression during the active myelination period of CNS development. Results presented in this dissertation demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE functions in conjunction with a heterologous thymidine kinase promoter. However, the relative location of the ASE appears to be important. When placed upstream of the 2.4 kb Plp 5'-flanking DNA in Plp-lacZ constructs, or downstream of the lacZ expression cassette, the ASE was no longer active. Thus, the ASE may need to be within the context of the intron in order to function. The orthologous sequence from rat functionally substituted for the mouse ASE in Plp-lacZ constructs, unlike those from human, cow or pig. This information can be used to help discern critical nucleotides within the ASE sequence, necessary for its function. To define the essential functions of the ASE, targeted mutagenesis in the mouse was performed to delete the ASE from its normal chromosomal context. Results presented here suggest that the ASE is necessary but not sufficient to govern the increase in Plp gene activity observed during the active myelination period of CNS development. In fact, deletion of the ASE seems to have a more potent effect earlier on during brain development. However, the ASE deleted mice (PlpNeoDeltaASE) have a "floxed" Neo gene cassette in place of the ASE, which may cause interference. Therefore, future studies will address the essential functions of the ASE in mice that no longer contain the positive selection cassette by crossing the PlpNeoDeltaASE mice with the Ella-cre deleter strain.