Epigenetic Regulation of Sox4 during Palate Development

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, identified Sox4 as a differentially expressed gene. In this study, we have examined if the differential expression of Sox4 in the palate is due to changes in DNA methylation. Materials & methods In situ hybridization analysis was used to localize the expression of Sox4 in the developing murine secondary palate. CpG methylation profiling of a 1.8-kb upstream region of Sox4 in the secondary palate from GD12–14 and transfection analysis in murine embryonic maxillary mesenchymal cells using Sox4 deletion, mutant and in vitro methylated plasmid constructs were used to identify critical CpG residues regulating Sox4 expression in the palate. Results Spatiotemporal analysis revealed that Sox4 is expressed in the medial edge epithelium and presumptive rugae-forming regions of the palate from GD12 to GD13. Following palatal shelf fusion on GD14, Sox4 was 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 of Sox4, 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 in Sox4 mRNA expression. Changes in the methylation of DMR4 were attributed primarily to CpGs 83 and 85. Conclusion Our studies indicate that Sox4 is 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.

Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk

“…Several observations support this hypothesis. First, multiple studies suggest a critical role for DNA methylation changes in normal murine palatogenesis [12,13] as well as in murine models of isolated orofacial clefts [14,15]. Second, like folate, increased maternal intake of choline has been associated with decreased CL/P risk [16].…”

Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure inDrosophila

“…, 2011 ). Comparable morphogenic movements are conserved across phylogeny and include vertebrate cell sheet movements that lead to neural tube and palate formation ( Copp and Greene, 2013 ; Seelan et al. , 2013 ; Martin and Goldstein, 2014 ; Razzell et al.…”

Nucleic acid methylation and orofacial morphogenesis

“…Sox4 , identified as a differentially expressed gene during murine secondary palate development (Mukhopadhyay, Greene, & Pisano, ), is a key signaling molecule located at a nodal point where several developmental pathways (e.g., TGF‐β, Wnt, and Hippo signaling pathways) critical for secondary palate development converge. CpG methylation profiling identified two CpG residues in the putative Sox4 promoter whose methylation levels correlated well with mRNA expression during palatal ontogeny (Seelan, Mukhopadhyay, et al, ). These residues were found, by functional studies, to be essential for Sox4 expression in the embryonic secondary palate.…”

“…These residues were found, by functional studies, to be essential for Sox4 expression in the embryonic secondary palate. Sox4 may be involved in palatal fusion, palatal extension, and/or the maintenance of the neural stem cell niche in the rugae (Seelan, Mukhopadhyay, et al, ). It is worth noting that SOX4 has been considered a strong candidate for human nonsyndromic CLP (Juriloff & Harris, ).…”