Stringent comparative sequence analysis reveals SOX10 as a putative inhibitor of glial cell differentiation

Abstract

BackgroundThe transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination.ResultsWe developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function.ConclusionsOur computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the computational and functional datasets we present here will be valuable for the study of transcriptional regulation, SOX protein function, and glial cell biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3167-3) contains supplementary material, which is available to authorized users.

A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve

“…1 ). First, the human (hg18), mouse (mm9), and chicken (Gal3) genomes were downloaded from the UCSC Genome Browser [ 23 ], aligned using MultiPipMaker [ 24 ], and the alignments analyzed using ExactPlus [ 25 ] to identify genomic segments that are identical among the three species and at least five base pairs in length [ 26 ]. Next, conserved regions overlapping a SNP that was ‘validated by-frequency’ [i.e., SNPs that were submitted (dbSNP 130) with allele frequency information] were identified to generate a panel of conserved regions harboring a SNP.…”

“…We then performed RNA-Seq analysis in an unbiased attempt to identify candidate target genes with altered expression. The rSOX-4 element is a 651-base-pair region that resides in a ~ 1 Mb interval devoid of annotated genes (Additional file 8 : Figure S5A) and that displayed genomic features indicating that it is an active, SOX10-bound enhancer, including: H3K27 acetylation from adult rat peripheral nerve [ 43 ], SOX10 ChIP-Seq from P15 rat sciatic nerve [ 39 ], and DNase hypersensitivity from the S16 cell line [ 26 ] (Additional file 8 : Figure S5A). Briefly, repair templates were generated with ~ 1 Kb arms of homology to the 5′ and 3′ regions surrounding the 651-base-pair rSOX-4 element, and flanking a floxed blasticidin (or neomycin) resistance cassette (Fig.…”

“…The computational and functional studies described here yielded a large panel of SNPs that reside in highly conserved regions, RNA-Seq data from cultured Schwann cells, and small panels of regulatory SNPs (rSNPs) and cis -regulatory elements (CREs) useful for investigators studying peripheral nerve and SOX10 biology. Furthermore, the pipeline developed here is flexible and may accommodate additional CRE information when it becomes available; for example, we combined our pipeline with previously unavailable SOX10 ChIP-Seq [ 39 ], S16 DNase hypersensitivity [ 26 ], and H3K27 acetylation [ 43 ] datasets to prioritize putative SOX10 binding sites for functional validation. There are, however, several inherent limitations to our approach that could be improved upon.…”

“…Indeed, our efforts suggest that the employment of strict conservation analyses does not dramatically enrich for functional regulatory elements and associated rSNPs. To address this problem, one could overlap available SOX10 ChIP-Seq data [ 39 ] with our data set of SOX10 consensus sequences [ 26 ] and study SNPs that map to overlapping genomic sequences, regardless of sequence conservation. Second, we tested a small subset of the 6164 regions predicted by our computational approach.…”

Waardenburg syndrome: a rare cause of inherited neuropathy due to SOX10 mutation

“…SOX10, one of the myelin‐specific transcription factors, displays activity as a transcriptional activator (Kuhlbrodt et al, ) through binding to the promoters and/or enhancers of target genes in cooperation with other transcription factors. SOX10 and EGR2 synergistically regulate the expression of key myelin‐related genes (Gopinath et al, ) . SOX10 and EGR2 directly bind to the Connexin 32 promoter activating Connexin 32 expression (Bondurand et al, ) .…”