Restricted accessResearch articleFirst published online 2018-06
Genome-Wide DNA Methylation Analysis During Palatal Fusion Reveals the Potential Mechanism of Enhancer Methylation Regulating Epithelial Mesenchyme Transformation
Epithelial mesenchyme transformation (EMT) of the medial edge epithelium (MEE) is the crucial process during palatal fusion. This work is aimed to elucidate the enhancer regulatory mechanism by genome-wide DNA methylation analysis of EMT during palatal fusion. Over 800 million clean reads, 325 million enzyme reads, and 234 million mapping reads were generated. The mapping rate was 68.85–74.32%, which included differentially methylated 17299 CCGG sites and 2363 CCWGG sites (p < 0.05, log2FC >1). Methylated sites in intron and intergenic regions were more compared to other regions of all DNA elements. GO and KEGG analysis indicated that differential methylation sites related to embryonic palatal fusion genes (HDAC4, TCF7L2, and PDGFRB) at the enhancer were located on CCWGG region of non-CpG islands. In addition, the results showed that the enhancer for HDAC4 was hypermethylated, whereas the enhancers for TCF7L2 and PDGFRB were hypomethylated. The methylation status of enhancer regions of HDAC4, PDGFRB, and TCF7L2, involved in the regulation of the EMT during palatal fusion, may enlighten the development of novel epigenetic biomarkers in the treatment of cleft palate.
Get full access to this article
View all access options for this article.
References
1.
AckermansM., ZhouH., CarelsC., WagenerF., and Von den HoffJ. (2011). Vitamin A and clefting: putative biological mechanisms. Nutr Rev, 69, 613–624.
2.
AnderssonR., GebhardC., Miguel-EscaladaI., HoofI., BornholdtJ., BoydM., et al. (2014). An atlas of active enhancers across human cell types and tissues. Nature, 507, 455–461.
3.
AnderssonR., SandelinA., and DankoC. (2015). A unified architecture of transcriptional regulatory elements. Trends Genet, 31, 426–433.
4.
AranD., and HellmanA. (2013). DNA methylation of transcriptional enhancers and cancer predisposition. Cell, 154, 11–13.
5.
AranD., SabatoS., and HellmanA. (2013). DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes. Genome Biol, 14, R21.
6.
BarresR., OslerM.E., YanJ., RuneA., FritzT., CaidahlK., et al. (2009). Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Cell Metab, 10, 189–198.
7.
BogdanovićO., and VeenstraG. (2009). DNA methylation and methyl-CpG binding proteins: developmental requirements and function. Chromosoma, 118, 549–565.
8.
BurgersW., FuksF., and KouzaridesT. (2002). DNA methyltransferases get connected to chromatin. Trends Genet, 18, 275–277.
9.
CaiafaP., and ZampieriM. (2005). DNA methylation and chromatin structure: the puzzling CpG islands. J Cell Biochem, 94, 257–265.
10.
CampbellJ., SmithM., FisherJ., and WarrenD. (2004). Dose-response for retinoic acid-induced forelimb malformations and cleft palate: a comparison of computerized image analysis and visual inspection. Birth Defects Res B Dev Reprod Toxicol, 71, 289–295.
11.
CingolaniP., PlattsA., Wang leL., CoonM., NguyenT., WangL., et al. (2012). A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly, 6, 80–92.
12.
Cohen-KarniD., XuD., AponeL., FomenkovA., SunZ., DavisP.J., et al. (2011). The MspJI family of modification-dependent restriction endonucleases for epigenetic studies. Proc Natl Acad Sci U S A, 108, 11040–11045.
13.
CuervoR., ValenciaC., ChandraratnaR., and CovarrubiasL. (2002). Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid. Dev Biol, 245, 145–156.
14.
DeatonA.M., and BirdA. (2011). CpG islands and the regulation of transcription. Genes Dev, 25, 1010–1022.
15.
EssaouiM., NizonM., BeaujardM.P., CarrierA., TantauJ., de BloisM.C., et al. (2013). Monozygotic twins discordant for 18q21.2qter deletion detected by array CGH in amniotic fluid. Eur J Med Genet, 56, 502–505.
16.
FeldmannA., IvanekR., MurrR., GaidatzisD., BurgerL., and SchubelerD. (2013). Transcription factor occupancy can mediate active turnover of DNA methylation at regulatory regions. PLoS Genet, 9, e1003994.
17.
GeimanT.M., and RobertsonK.D. (2002). Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together?. J Cell Biochem, 87, 117–125.
18.
HaberlandM., MontgomeryR.L., and OlsonE.N. (2009). The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet, 10, 32–42.
19.
JaenischR., and BirdA. (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet, 33Suppl, 245–254.
20.
JinT., George FantusI., and SunJ. (2008). Wnt and beyond Wnt: multiple mechanisms control the transcriptional property of beta-catenin. Cell Signal, 20, 1697–1704.
21.
KafriT., ArielM., BrandeisM., ShemerR., UrvenL., McCarreyJ., et al. (1992). Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev, 6, 705–714.
22.
KalluriR., and WeinbergR. (2009). The basics of epithelial-mesenchymal transition. J Clin Invest, 119, 1420–1428.
23.
Kieffer-KwonK.R., TangZ., MatheE., QianJ., SungM.H., LiG., et al. (2013). Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation. Cell, 155, 1507–1520.
24.
KimT., and ShiekhattarR. (2015). Architectural and functional commonalities between enhancers and promoters. Cell, 162, 948–959.
25.
KlinghofferR.A., HamiltonT.G., HochR., and SorianoP. (2002). An allelic series at the PDGFalphaR locus indicates unequal contributions of distinct signaling pathways during development. Dev Cell, 2, 103–113.
26.
LawJ.A., and JacobsenS.E. (2010). Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet, 11, 204–220.
27.
LevinH.L., and MoranJ.V. (2011). Dynamic interactions between transposable elements and their hosts. Nat Rev Genet, 12, 615–627.
28.
LiE., BestorT., and JaenischR. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69, 915–926.
29.
LiR., YuC., LiY., LamT.W., YiuS.M., KristiansenK., et al. (2009). SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics (Oxford, England), 25, 1966–1967.
LoenarzC., GeW., ColemanM., RoseN., CooperC., KloseR., et al. (2010). PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nepsilon-dimethyl lysine demethylase. Hum Mol Genet, 19, 217–222.
32.
MacDonaldB.T., TamaiK., and HeX. (2009). Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell, 17, 9–26.
33.
McCarthyN., LiuJ.S., RicharteA.M., EskiocakB., LovelyC.B., TallquistM.D., et al. (2016), Pdgfra and Pdgfrb genetically interact during craniofacial development. Dev Dyn, 245, 641–652.
34.
McGinnisS., and MaddenT.L. (2004). BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res, 32, W20–W25.
35.
NawshadA. (2008). Palatal seam disintegration: to die or not to die?. that is no longer the question. Dev Dyn, 237, 2643–2656.
36.
OhganeJ., WakayamaT., KogoY., SendaS., HattoriN., TanakaS., et al. (2001). DNA methylation variation in cloned mice. Genesis, 30, 45–50.
37.
PetersJ. (2014). The role of genomic imprinting in biology and disease: an expanding view. Nat Rev Genet, 15, 517–530.
38.
PetrascheckM., EscherD., MahmoudiT., VerrijzerC.P., SchaffnerW., BarberisA. (2005). DNA looping induced by a transcriptional enhancer in vivo. Nucleic Acids Res, 33, 3743–3750.
39.
QinF., ShenZ., PengL., WuR., HuX., ZhangG., et al. (2014). Metabolic characterization of all-trans-retinoic acid (ATRA)-induced craniofacial development of murine embryos using in vivo proton magnetic resonance spectroscopy. PLoS One, 9, e96010.
40.
QuinlanA.R., and HallI.M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics (Oxford, England), 26, 841–842.
41.
RahimovF., JugessurA., and MurrayJ.C. (2012). Genetics of nonsyndromic orofacial clefts. Cleft Palate Craniofac J, 49, 73–91.
42.
RiceD.P. (2005). Craniofacial anomalies: from development to molecular pathogenesis. Curr Mol Med, 5, 699–722.
43.
RobinsonM.D., McCarthyD.J., and SmythG.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics (Oxford, England), 26, 139–140.
44.
SaluzH.P., JiricnyJ., and JostJ.P. (1986). Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc Natl Acad Sci U S A, 83, 7167–7171.
45.
ShlyuevaD., StampfelG., and StarkA. (2014). Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet, 15, 272–286.
StuppiaL., CapogrecoM., MarzoG., La RovereD., AntonucciI., GattaV., et al. (2011). Genetics of syndromic and nonsyndromic cleft lip and palate. J Craniofac Surg, 22, 1722–1726.
48.
SunJ., WangD., and JinT. (2010). Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells. Biochim Biophys Acta, 1800, 344–351.
49.
ThieryJ., AcloqueH., HuangR., and NietoM. (2009). Epithelial-mesenchymal transitions in development and disease. Cell, 139, 871–890.
VegaR.B., MatsudaK., OhJ., BarbosaA.C., YangX., MeadowsE., et al. (2004). Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell, 119, 555–566.
52.
VernimmenD., and BickmoreW. (2015). The hierarchy of transcriptional activation: from enhancer to promoter. Trends Genet, 31, 696–708.
53.
WangS., LvJ., ZhangL., DouJ., SunY., LiX., et al. (2015). MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes. Open Biol, 5, pii: 150130.
54.
ZhangJ., KobertK., FlouriT., and StamatakisA. (2014). PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics (Oxford, England), 30, 614–620.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
