Schwann cells of the peripheral nervous system require coordinated gene expression changes for myelin development to occur in a timely fashion. While epigenetic mechanisms play a significant role in a variety of developmental processes, distinct chromatin remodeling pathways that modulate gene expression changes during peripheral myelination have only recently begun to be investigated. Previous studies uncovered a novel mechanism for NAB repression of Egr2 target genes through interaction with the Chd4 subunit of the NuRD complex (Srinivasan et al., 2006
; Mager et al., 2008
). Our results now demonstrate that Chd4 ablation in the peripheral nervous system results in impaired Schwann cell differentiation leading to a developmental delay in myelination, reflected in the prevalence of pro-myelinating Schwann cells, hypomyelination, and enhanced proliferation at P8. Inefficient initiation of the myelin producing stage in Schwann cell development indicates loss of Chd4 results in delayed and incomplete Schwann cell differentiation. The myelination status deteriorates considerably by P30, resulting in significant ataxia, myelin degeneration, and altered gene expression patterns, indicating that Chd4 may also play a role during later stages of Schwann cell myelination for the maturation and homeostasis of the myelin sheath beyond P15.
Although the association of Chd4 with NAB coregulators provided the mechanistic basis for this study, the resulting phenotype is significantly less severe than that caused by deletion of both NAB genes, or by knock-in of a NAB-resistant allele of Egr2 (Le et al., 2005b
; Desmazieres et al., 2008
; Baloh et al., 2009
). One possible explanation could be redundancy with Chd3. Chd4 is highly similar to Chd3, and both proteins have been shown to be part of the NuRD complex (Ramírez and Hagman, 2009
; Wang et al., 2009a
) and have been shown to interact with Nab2 (Srinivasan et al., 2006
). However, using an antibody that detects both Chd3 and Chd4, there seems to be little background expression of Chd3 after deletion of Chd4, although it is possible that residual levels could compensate to some degree. In addition, NAB proteins have two independent repression domains, one of which exerts its activity in an Hdac- and Chd4-independent manner (Srinivasan et al., 2006
). Therefore, it is anticipated that NAB proteins retain some level of repressive activity in the absence of NuRD activity.
Our studies of Chd4-deficient nerve revealed not only derepression of genes that are normally repressed in mature myelinatng Schwann cells, but also reduced levels of several critical myelin genes in sciatic nerves from knockout mice. Accordingly, ChIP analysis in myelinating sciatic nerve detected binding of Chd4 and another NuRD subunit (Mta2) at promoters of repressed genes, consistent with direct repression of negative regulators by the NuRD complex during development. This analysis also detected binding of Chd4 and MTA2 to promoters that are induced during myelination. Our findings are similar in many respects to analysis of Nab1/Nab2 double knockout mice (Le et al., 2005b
), which revealed that Nab proteins are required for both gene activation and gene repression by Egr2.
Other studies of Chd4 function have shown direct requirement of the NuRD complex to both activate and repress transcription in hematopoietic development (Yoshida et al., 2008
; Miccio and Blobel, 2010
; Miccio et al., 2010
). Although we detect binding of NuRD components at both activated and repressed genes, it is possible that H3K4 trimethylation at active promoters prevents repressive remodeling by the NuRD complex (Nishioka et al., 2002
; Zegerman et al., 2002
). In addition, the activation state of a given promoter may reflect a balance between deacetylation by the NuRD complex, and acetylation by other coactivators (CBP/p300) which are recruited by Egr2 and associated factors. Interestingly, genomic profiling of Hdac binding has revealed widespread binding of Hdac1/2 to active genes (Wang et al., 2009b
). Therefore, it is possible that histone deacetylation at regulatory regions may precede positive or negative methylation (K4 vs. K9/K27) at different genes as myelination proceeds.
Recent studies have shown that peripheral nerve myelination depends upon activity of Hdac1/2 (Chen et al., 2011
; Jacob et al., 2011
). The role of these histone deacetylases is mechanistically complex since they are constituents of multiple complexes (NuRD, Sin3a, CoRest). In addition, it is clear that they also have important non-histone substrates. For example, in glial development, Hdac1 and Hdac2 regulate oligodendrocyte differentiation by disrupting the beta-catenin-TCF interaction (Ye et al., 2009
), and HDAC-mediated deacetylation of NF-κB is a determinant of gene regulation (Chen et al., 2011
). The phenotype of the Chd4-deficient peripheral nerve is significantly milder compared to Hdac1/2 deficient mice, which exhibit failure to myelinate and lack of Egr2 expression (Chen et al., 2011
; Jacob et al., 2011
). We did not observe any obvious level of apoptosis exhibited by Hdac-deficient mice (Jacob et al., 2011
), and the level of Oct6 expression indicates that NF-kB acetylation status is likely not perturbed in Chd4-deficient mice. In addition, Western blotting indicated no significant difference in β-catenin at P30. This likely reflects the fact that NuRD complexes mediate only a subset of Hdac-regulated events.
Substantial progress has been made in identifying transcription factors that coordinate gene expression during myelination. However, for many such studies, a common feature of the knockout phenotype is reduced Krox20/Egr2 expression, as observed in the Sox10, Calcineurin B1, Hdac, Oct6, dicer and YY1 knockout studies of peripheral nerve (Blanchard et al., 1996
; Kao et al., 2009
; Finzsch et al., 2010
; He et al., 2010
; Pereira et al., 2010
; Yun et al., 2010
; Chen et al., 2011
; Jacob et al., 2011
). Importantly, the Chd4 knockout phenotype does not stem from reduced Egr2 expression, suggesting that gene regulation by Chd4 is required downstream of (or parallel to) Egr2 activity.
While development favors induction of a myelinated nerve state, these conditions must be quickly reversible in response to nerve injury. During nerve injury, Schwann cells undergo a process of dedifferentiation, and suppress myelin genes while inducing negative regulators (Jessen and Mirsky, 2008
). For the nervous system to complete a full recovery and remyelinate, the balance of transcriptional control must then swing back to promoting Schwann cell myelination. Ineffective suppression of negative regulators significantly affects both timing and extent of remyelination. The molecular pathways controlling repression of dedifferentiation factors are not well understood. However, recent studies have elucidated the importance of epigenetic regulation of myelin gene expression. Shen et al. (2008) showed remyelination efficiency in oligodendrocytes decreases with age due to the reduced ability of senescent cells to recruit HDAC proteins to the promoters of differentiation inhibitors and neural stem cell markers. Of note, the NuRD complex is one of the major HDAC-containing chromatin-remodeling complexes. Moreover, subunits of the NuRD complex are prone to silencing during aging leading to changes in chromatin modification, structure, and protein recruitment (Pegoraro and Misteli, 2009
). Improving our understanding of the epigenetic mechanisms that control myelin formation and maintenance, including recruitment of chromatin remodeling complexes, will be critical to elucidate the genomic programming required for myelination.