Epigenetic regulation ofSox4during palate development

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Abstract

Aim:

Identification of genes that contribute to secondary palate development provide a better understanding of the etiology of palatal clefts. Gene-expression profiling of the murine palate from gestational days 12–14 (GD12–14), a critical period in palate development, identifiedSox4as a differentially expressed gene. In this study, we have examined if the differential expression ofSox4in the palate is due to changes in DNA methylation.

Materials & methods:

In situhybridization analysis was used to localize the expression ofSox4in the developing murine secondary palate. CpG methylation profiling of a 1.8-kb upstream region ofSox4in the secondary palate from GD12–14 and transfection analysis in murine embryonic maxillary mesenchymal cells usingSox4deletion, mutant andin vitromethylated plasmid constructs were used to identify critical CpG residues regulatingSox4expression in the palate.

Results:

Spatiotemporal analysis revealed thatSox4is expressed in the medial edge epithelium and presumptive rugae-forming regions of the palate from GD12 to GD13. Following palatal shelf fusion on GD14,Sox4was expressed exclusively in the epithelia of the palatal rugae, structures that serve as signaling centers for the anteroposterior extension of the palate, and that are thought to serve as neural stem cell niches. Methylation of a 1.8-kb region upstream ofSox4, containing the putative promoter, completely eliminated promoter activity. CpG methylation profiling of the 1.8-kb region identified a CpG-poor region (DMR4) that exhibited significant differential methylation during palate development, consistent with changes inSox4mRNA expression. Changes in the methylation of DMR4 were attributed primarily to CpGs 83 and 85.

Conclusion:

Our studies indicate thatSox4is an epigenetically regulated gene that likely integrates multiple signaling systems for mediating palatal fusion, palatal extension and/or the maintenance of the neural stem cell niche in the rugae.

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