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.

KEYWORDS::

• cleft palate
• CpG methylation
• epigenetics
• medial edge epithelium
• orofacial
• rugae
• secondary palate
• Sox4

Disclaimer

The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NCRR, NIH.

Financial & competing interests disclosure

This work was supported in part by NIH grants DE018215, HD053509 and P20 RR017702 from the COBRE program of the National Center for Research Resources and NIGMS, and by a grant from the Cleft Palate Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Notes

Bold indicates CpG sites.

Bold and underline indicate CpG sites of amplicons 3 and 4 exhibiting differential methylation.

>>3: Start of amplicon 3; >>4: Start of amplicon 4; 3<<: End of amplicon 3; 4<<: End of amplicon 4.

Additional information

Funding

This work was supported in part by NIH grants DE018215, HD053509 and P20 RR017702 from the COBRE program of the National Center for Research Resources and NIGMS, and by a grant from the Cleft Palate Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.