FAIRE analysis showed that many peaks of Egr2 binding lie within areas of open chromatin, which generally correlates with regulatory regions (Giresi et al. 2007
; Giresi and Lieb 2009
). Interestingly, many of the peaks of Egr2 binding revealed by ChIP-chip analysis extended over several hundred basepairs (e.g. ), which may reflect some bridging interactions (through various cofactors) to adjacent DNA segments. In contrast, the FAIRE analysis often provided a more finely focused peak that could be used to more accurately pinpoint Egr2 sites.
There seems to be considerable heterogeneity in the location of Egr2 binding sites. The simplest case is that of the Connexin 32
gene, in which both Egr2 and Sox10 bind to the proximal promoter region of the regulated P2 promoter. Interestingly, Connexin 32
is the only myelin gene in which noncoding promoter mutations have been identified associated with human peripheral neuropathy (Bondurand et al. 2001
; Houlden et al. 2004
). In contrast, activation of other myelin genes such as Ndrg1
() and Mpz
(Jang and Svaren 2009
) is associated with multiple distributed Egr2 binding sites, including intragenic regions (often in conserved sites within introns). Based on similar analyses of other transcription factors, it is expected that not all sites are functionally required for regulation of specific genes in Schwann cells. It has been proposed that at least some binding sites may simply reflect the substantial affinity of transcription factors for DNA in general, but that functional sites are bound at quantitatively higher levels (Li et al. 2008
). Accordingly, a strong site in the Ndrg1 gene was more highly activated in our tranfection assay compared to another intragenic site with a lower signal in our quantitative PCR validation. However, it is possible that weaker binding sites may still contribute to gene activation in the context of the whole locus.
The ChIP-chip analysis showed that Egr2 binds to the promoters of many of the lipid biosynthesis genes included on this array. Previous studies have indicated that the major regulators for lipid biosynthetic genes—sterol regulatory element binding proteins (SREBPs)—are required for efficient myelination (Verheijen et al. 2003
; Verheijen et al. 2009
). Since SREBPs generally bind to proximal promoter regions of their target genes (Seo et al. 2009
), this may indicate that Egr2 interacts with SREBP factors in these promoters. Consistent with this, some lipid biosynthetic genes are synergistically activated by Egr2 and SREBPs in transfection experiments (LeBlanc et al. 2005
). Egr2 has a binding specificity that overlaps to some extent with that of Sp1, which has been reported to bind adjacent to SREBPs on the promoters of many genes involved in lipid metabolism (reviewed in Shimano 2001
). Therefore, it is possible that Egr2 utilizes some previously defined Sp1 binding sites to augment the activation of lipid biosynthetic genes to the high levels required for myelination.
Tissue-specific expression of myelin genes in Schwann cells depends upon combinatorial interactions between transcription factors. For example, both Egr2 and Sox10 are present in tissues that do not express myelin genes (e.g. T cells and melanocytes, respectively). Therefore, expression of either of these factors alone is not sufficient for high level expression of myelin genes. We had proposed that a cluster of conserved Egr2 and Sox10 dimeric sites may be a common theme in regulatory elements of developmentally regulated myelin genes in Schwann cells (Jones et al. 2007
). The ChIP-chip analysis revealed extensive colocalization of Egr2 with Sox10, and binding site analysis identified putative binding sites for Sox10 in most Egr2 binding peaks. The extensive colocalization of Egr2 with Sox10 at various sites may reflect direct interaction of the two factors (Wissmuller et al. 2006
; Jones et al. 2007
; LeBlanc et al. 2007
Many Egr2 target genes are dynamically induced during myelination since they are required for the formation of myelin structure. On the other hand, some genes are shown to be downregulated as myelination proceeds, including Sox2, Id2, Id4,
; Shy et al. 1996
; Verheijen et al. 2003
; Le et al. 2005a
). Based on the elevated levels of these genes in peripheral nerve of Egr2- and NAB-deficient mice, it has been proposed that Egr2 and associated NAB corepressors downregulate their expression (Zorick et al. 1999
; Le et al. 2005a
; Le et al. 2005b
; Decker et al. 2006
; Mager et al. 2008
). Our previous work showed that Egr2 and its corepressors, Nab1 and Nab2, bind to the Id2
gene in a developmentally-regulated manner as it is being repressed in peripheral nerve (Mager et al. 2008
). The ChIP-chip analysis extends these findings by showing Egr2 binds to the promoters of virtually all of the genes (included on our array) that decline as myelination proceeds. It has not been determined how Egr2/Nab complexes repress some genes, while Egr2 activates myelin genes. Interestingly, Sox10 binding was also observed in repressed genes. Sox10 has been shown to be sumoylated on several conserved lysines, which is generally correlated with gene repression (Taylor and Labonne 2005
; Girard and Goossens 2006
), although further analysis will be required to determine if this plays a role in gene repression during myelination.
Overall, genome-wide mapping of Egr2 binding using ChIP-chip (or ChIP-Seq) in a physiologically relevant system will help elucidate the cognate elements that mediate gene regulation by Egr2 during PNS myelination. Moreover, it is expected that combining such studies with analyses of open chromatin (by DNaseI or FAIRE), cross-species sequence conservation, and colocalization with other transcription factors will accelerate discovery of the regulatory elements required for the developmental regulation of myelination.