Functional analysis of myelin basic protein gene regulation

Through this investigation I hoped to illuminate and characterize any combinatorial relationships that might exist amongst the regulatory sequences that drive expression of the myelin basic protein gene. With such insight, I expected to learn more of the mechanism/s regulating myelin sheath formation, maintenance and repair. In the search for regulatory regions within the mbp gene, four highly conserved non-protein-coding modules were found in its 5' flanking sequence. In the context of reporter constructs in transgenic mice, these regulatory modules confer distinct cell specificity and developmental expression programs. M1 and M3 drive expression in oligodendrocytes while M4 drives Schwann cell expression. However, the expression programs realized by these reporter constructs also exposed higher levels of regulatory organization; e.g., when M3 is dissociated from neighboring flanking sequences it acquires the ability to drive transient expression in Schwann cells.;Based on the expression programming of reporter genes, M3 is critical for achieving high levels of mbp expression in myelinating oligodendrocytes. To determine if the expression phenotypes of reporter genes accurately reflected the activity of enhancers in the context of the endogenous locus, I used gene targeting to delete M3 in the endogenous mbp locus. This deletion had the anticipated consequence on the mbp expression profile in both oligodendrocytes and Schwann cells and in turn, led to abnormal quantitative and qualitative myelin phenotypes in the CNS bringing disease relevance to my observations.;Finally, mice bearing the mbp allele deleted of M3 were investigated for the accumulated levels of golli transcripts. These transcripts originate from a promoter located 70Kb upstream of mbp, include different combinations of golli and mbp exons and are expressed widely in the nervous and immune systems of developing and mature mice. My investigation revealed that M3 is a multi-functional enhancer controlling both mbp and golli transcription and I show that its golli related activity plays an important role in the process of myelination.;To further understand how the four known mbp regulatory modules orchestrate the expression of the mbp gene, I first generated reporter constructs designed to contain most module combinations. These were inserted in single copy in a common orientation at a common site 5' of the HPRT locus and their qualitative and quantitative regulatory potential was analyzed.