Our laboratory has focused our longstanding interest in chromatin regulation and gene expression on elucidating the transcriptional control of the myelination process. Myelination is a vital component of nervous system function, and is adversely affected in a variety of diseases such as Charcot-Marie-Tooth Disease and Multiple Sclerosis. Our studies have so far focused on myelination by Schwann cells in the peripheral nervous system. Many features of this process make it an excellent system for developmental analysis of epigenetic and transcriptional mechanisms in vivo. Large peripheral nerves are highly enriched in Schwann cells, which comprise approximately 80-90% of the tissue. Of these, >50% of these cells are myelinating Schwann cells. In addition, the presence of neuronal nuclei in the spinal cord allows physical separation from Schwann cell nuclei in peripheral nerve. In contrast, analysis of CNS is more complicated because of the more heterogeneous array of cell types present. The developmental regulation of myelination occurs largely postnatally, and there is sufficient material for chromatin immunoprecipitation analysis at early (P0-P5), and later stages of the myelination process. Based on these unique features, ChIP techniques applied to peripheral nerve provide a tremendous opportunity to analyze developmentally regulated epigenetic changes within a physiological system. Ultimately, our studies are designed to construct an integrated map of transcriptional and epigenetic factors that control the initiation, progression, and maintenance of myelination.
Transcriptional Regulation by Egr2 and Sox10. Our studies have identified a number of regulatory elements in major myelin genes, such as Peripheral Myelin Protein (Pmp22) and Myelin Protein Zero (Mpz), which are coordinately regulated by Egr and Sox10. The HMG box transcription factor, Sox10, is required for Schwann cell specification and continues to be expressed throughout adulthood. Egr2 (also known as Krox20) is induced at the onset of myelination by axonal signals, and is also required for initiation and maintenance of myelination. To obtain a more comprehensive view of the action of these two transcription factors, we have obtained genome-wide data on Egr2 and Sox10 binding, which have shown extensive colocalization, but there also unique sets of target genes controlled by the two factors.
Chromatin Remodeling during Myelination. The regulation of myelination by Egr2 requires interaction with NAB (NAB1 and NAB2) corepressors, and there is evidence that Egr2 causes not only gene activation but also gene repression. Based on our identification of an interaction of NAB proteins with the NuRD (Nucleosome Remodeling and Deacetylase) complex, we have performed a Schwann cell-specific deletion of the core Chd4 subunit of this complex. The resulting phenotype indicates that the NuRD complex is required for timely and stable myelination in peripheral nerve, and we are currently characterizing the function of the NuRD complex in both gene activation and repression.
Integrated Regulation of myelination by miRNAs. Our studies of transcriptional regulation have uncovered a set of miRNAs that are dependent upon Sox10 regulation. Some of the miRNAs (miR338) have been shown to be involved in oligodendrocyte regulation, and others are involved in regulation of proliferation. Since miRNAs are required for successful myelination, we are interested in determining the interactions of miRNA-mediated regulation with the transcriptional networks regulated by Egr2 and Sox10.
Regulation of Pmp22. Duplication of a 1.4 Mb chromosomal segment containing the PMP22 gene is the most common cause of Charcot-Marie-Tooth Disease (classified as CMT1A). In a project funded by the Charcot-Marie-Tooth Association (CMTA), we have been mapping the regulatory elements of the PMP22 gene. Using both analysis of Egr2/Sox10 binding and also analysis of open chromatin regions by FAIRE (Formaldehyde-assisted identification of regulatory elements), we have identified several new regulatory elements. Moreover, we are using this information to develop new assays that can be used to screen compound libraries at the NIH Chemical Genomics Center in order to identify drugs that lower Pmp22 expression and treat the root cause of CMT1A.