Abstract
Objective:Cleft palate (CP) is a congenital birth defect caused by the failure of palatal fusion. Little is known about the potential role of DNA methylation in the pathogenesis of CP. This study aimed to explore the potential role of DNA methylation in the mechanism of CP.Methodology:We established an all-trans retinoic acid (ATRA)-induced CP model in C57BL/6J mice and used methylation-dependent restriction enzymes (MethylRAD, FspEI) combined with high-throughput sequencing (HiSeq X Ten) to compare genome-wide DNA methylation profiles of embryonic mouse palatal tissues, between embryos from ATRA-treated vs. untreated mice, at embryonic gestation day 14.5 (E14.5) (n=3 per group). To confirm differentially methylated levels of susceptible genes, real-time quantitative PCR (qPCR) was used to correlate expression of differentially methylated genes related to CP.Results:We identified 196 differentially methylated genes, including 17,298 differentially methylated CCGG sites between ATRA-treated vs. untreated embryonic mouse palatal tissues (P<0.05, log2FC>1). The CP-related genes Fgf16 (P=0.008, log2FC=1.13) and Tbx22 (P=0.011, log2FC=1.64,) were hypermethylated. Analysis of Fgf16 and Tbx22, using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), identified 3 GO terms and 1 KEGG pathway functionally related to palatal fusion. The qPCR showed that changes in expression level negatively correlated with methylation levels.Conclusions:Taken together, these results suggest that hypermethylation of Fgf16 and Tbx22 is associated with decreased gene expression, which might be responsible for developmental failure of palatal fusion, eventually resulting in the formation of CP.
Highlights
Cleft palate (CP) is a congenital birth defect caused by both environmental and genetic factors.1 It is universally acknowledged that palatal fusion is the most crucial process in the palate formation
The DNA methylation pattern of a mouse undergoes dynamic and widespread alterations during palatogenesis, and failing to establish correct methylation patterns can result in CP,6 which suggests that CP-susceptible genes (e.g. Fgf16 and Tbx22) in embryonic mice provide new clues to epigenetic markers involved in CP
In palate shelf tissue and histological sections of untreated E14.5 embryos, it can be observed that the palatal shelf has already contacted the midline and has fused to form the midline epithelial seam (MES) in the mid-anterior region, whereas in palate shelf tissue and histological sections from all-trans retinoic acid (ATRA)-treated embryos, the palatal shelf remained completely separated without fusion
Summary
Cleft palate (CP) is a congenital birth defect caused by both environmental and genetic factors. It is universally acknowledged that palatal fusion is the most crucial process in the palate formation. DNA methylation is an important epigenetic modification and plays a crucial role in many biological processes, such as embryogenesis, cellular differentiation, X-chromosome inactivation, genomic imprinting and transcriptional silencing.. Methylation patterns of specific genes have been found to undergo dynamic changes in embryonic development and contribute to tissue-specific gene expression.. The DNA methylation pattern of a mouse undergoes dynamic and widespread alterations during palatogenesis, and failing to establish correct methylation patterns can result in CP, which suggests that CP-susceptible genes (e.g. Fgf and Tbx22) in embryonic mice provide new clues to epigenetic markers involved in CP. Details on the methylation patterns of CP-susceptible genes during palatal fusion are very limited, and the methylation pattern of Fgf and Tbx underlying palate development and its contribution to CP is still unclear
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