| Summary: | Myelination is required for the faithful conduction of action potentials in the vertebrate nervous system. Oligodendrocytes are cells that produce myelin, a large extension of plasma membrane, which is composed of specialized lipids and proteins, and wrap neurons during myelination in the central nervous system (CNS). Our knowledge of myelin production, that is the production of specialized lipids and proteins in the myelin sheath, and myelination, how oligodendrocytes wrap neurons, however is incomplete particularly with respect to which transcription factors are required for myelin production. Recently our lab characterized a mouse mutant, hypomyelinated in the central nervous system, which was mapped to a disruption in the gene zinc-finger protein 191 (Zfp191). Characterization of a Zfp191 deficient mouse (Zfp191null) has previously been shown that the absence of ZFP191 results in hypomyelination.
ZFP191 belongs to a protein family that is well characterized for its role in transcriptional modulation. The transcriptional program that controls oligodendrocyte maturation and CNS myelination has not been fully characterized. Our previous analysis of the Zfp191null mouse demonstrated that a normal number of oligodendrocyte precursor cells trafficked correctly and displayed immunohistochemical reactivity for markers associated with proper maturation into oligodendrocytes. However, these oligodendrocytes had reduced levels of myelin basic protein (MBP), a required factor for myelin, and animals lacking ZFP191 did not possess myelinated neurons in the CNS. To explore how loss of ZFP191 results in hypomyelination I examined the role of ZFP191 in modulating the transcriptome and potential mechanisms for how ZFP191 functions in this potential modulation. I hypothesized that ZFP191 is functioning to modulate the transcription of key components that control myelin production and myelination.
Previous work has shown that the loss of ZFP191 results in decreased expression of key myelin related transcript levels in the whole brain when compared to a wild-type animal. Using massively parallel sequencing I examined how the loss of ZFP191 modulates the transcriptome of the whole brain, oligodendrocyte precursor cells and mature oligodendrocytes. The transcriptome could also have been perturbed due to the loss of myelin therefore I also examined the similar transcriptomes in another model of hypomyelination. This other model of hypomyelination is the result of the loss of MBP, which has previously shown is diminished in Zfp191null mice. The analysis described in Chapter 2 will help to parse out the role of ZFP191 in hypomyelination and what changes are and are not independent of the loss of MBP.
We currently know that loss of ZFP191 results in hypomyelination, but how this loss results in this phenotype is unclear. ZFP191 belongs to a family of proteins that are known to play key roles in transcriptional regulation. In Chapter 3 I utilized an unbiased approach to interrogate where ZFP191 is interacting at the genomic level. Using this methodology I also determined a ZFP191 DNA binding sequence. Sequences that were found were used in biochemical assays to confirm this interaction and the role of ZFP191 in transcriptional regulation.
Knowing how loss of ZFP191 disrupts the transcriptome is critical to understanding the underlying transcriptional networks that play a role in oligodendrocyte development and myelination. When transcriptome data was combined with locations in the genome where ZFP191 bound I began to determine the primary downstream targets of ZFP191. Chapter 4 compares the perturbed transcriptomes characterized in Chapter 2 with the identified ZFP191 binding sites in Chapter 3.
Chapter 5 synthesizes our current understanding of the role of ZFP191 and its role in oligodendrocytes. I present a model of how ZFP191 may affect transcriptional regulation in the oligodendrocyte. I also propose potential avenues of further experimentation and analysis that will deepen and clarify our understanding of oligodendrocyte maturation and myelination.
The Appendix includes supplemental files, available online, for primers used in my experiments and also the processed files for all of the parallel sequencing files used in Chapters 2-4.
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