Limits...
DNA methylation is developmentally regulated for genes essential for cardiogenesis.

Chamberlain AA, Lin M, Lister RL, Maslov AA, Wang Y, Suzuki M, Wu B, Greally JM, Zheng D, Zhou B - J Am Heart Assoc (2014)

Bottom Line: Quantitative real-time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2).Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer.Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co-expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY (A.A.C., M.L., A.A.M., Y.W., M.S., B.W., J.M.G., D.Z.).

Show MeSH

Related in: MedlinePlus

Has2 expression is regulated by Dnmt3b. A, MassArray showing increased Has2 enhancer methylation at E14.5 (n=2). B, RT‐qPCR showing decreased Has2 expression at E14.5 (n=3). C, RNA in situ hybridization showing that Has2 expression is predominantly in the atrioventricular canal (avc), with less expression in the myocardium (myo) at E11.5; and the expression is diminished by E14.5. mv/tv, mitral/tricuspid valve; ivs, interventricular septum. D, IHC showing Dnmt3b is predominantly expressed in AVC at E11.5. E, X‐gal staining showing the Nfatc1‐Cre mediated LacZ expression in the AVC at E11.5. F, RT‐qPCR showing that deletion of Dnmt3b resulted in increased Has2 expression at E11.5 and E14.5 (n=3). Error bars represent standard error. *Marks statistical significance (P<0.001, 2 factor ANOVA in [F]). ANOVA indicates analysis of variance; CKO, conditional knockout; IHC, immunohistochemistry; RT‐qPCR, quantitative real‐time polymerase chain reaction; WT, wild type.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4309105&req=5

fig07: Has2 expression is regulated by Dnmt3b. A, MassArray showing increased Has2 enhancer methylation at E14.5 (n=2). B, RT‐qPCR showing decreased Has2 expression at E14.5 (n=3). C, RNA in situ hybridization showing that Has2 expression is predominantly in the atrioventricular canal (avc), with less expression in the myocardium (myo) at E11.5; and the expression is diminished by E14.5. mv/tv, mitral/tricuspid valve; ivs, interventricular septum. D, IHC showing Dnmt3b is predominantly expressed in AVC at E11.5. E, X‐gal staining showing the Nfatc1‐Cre mediated LacZ expression in the AVC at E11.5. F, RT‐qPCR showing that deletion of Dnmt3b resulted in increased Has2 expression at E11.5 and E14.5 (n=3). Error bars represent standard error. *Marks statistical significance (P<0.001, 2 factor ANOVA in [F]). ANOVA indicates analysis of variance; CKO, conditional knockout; IHC, immunohistochemistry; RT‐qPCR, quantitative real‐time polymerase chain reaction; WT, wild type.

Mentions: Previous knockout studies in mice have shown that Has2 is essential for development of cardiac valves and septa.51,56 Here, our DNA methylation analysis indicated that an ACGT site located within 1 kb of a previously determined enhancer of Has2, marked by enriched H3K27ac at E14.5,44 exhibited an increase in methylation as confirmed by Sequenom's MassArray (Figure 7A). Based on what is known about DNA methylation, we expected to see a decrease in Has2 expression at E14.5, with confirmation by qPCR analysis (Figure 7B). To further characterize its expression in the developing hearts, we carried out RNA in situ hybridization. The results showed that Has2 expression was predominately expressed in the endocardial cells and their mesenchymal progeny cells that form the primitive heart valves at E11.5, but its expression markedly diminished by E14.5 (Figure 7C), consistent with its function in endocardial to mesenchymal transformation around E11.5 for heart valve development.


DNA methylation is developmentally regulated for genes essential for cardiogenesis.

Chamberlain AA, Lin M, Lister RL, Maslov AA, Wang Y, Suzuki M, Wu B, Greally JM, Zheng D, Zhou B - J Am Heart Assoc (2014)

Has2 expression is regulated by Dnmt3b. A, MassArray showing increased Has2 enhancer methylation at E14.5 (n=2). B, RT‐qPCR showing decreased Has2 expression at E14.5 (n=3). C, RNA in situ hybridization showing that Has2 expression is predominantly in the atrioventricular canal (avc), with less expression in the myocardium (myo) at E11.5; and the expression is diminished by E14.5. mv/tv, mitral/tricuspid valve; ivs, interventricular septum. D, IHC showing Dnmt3b is predominantly expressed in AVC at E11.5. E, X‐gal staining showing the Nfatc1‐Cre mediated LacZ expression in the AVC at E11.5. F, RT‐qPCR showing that deletion of Dnmt3b resulted in increased Has2 expression at E11.5 and E14.5 (n=3). Error bars represent standard error. *Marks statistical significance (P<0.001, 2 factor ANOVA in [F]). ANOVA indicates analysis of variance; CKO, conditional knockout; IHC, immunohistochemistry; RT‐qPCR, quantitative real‐time polymerase chain reaction; WT, wild type.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309105&req=5

fig07: Has2 expression is regulated by Dnmt3b. A, MassArray showing increased Has2 enhancer methylation at E14.5 (n=2). B, RT‐qPCR showing decreased Has2 expression at E14.5 (n=3). C, RNA in situ hybridization showing that Has2 expression is predominantly in the atrioventricular canal (avc), with less expression in the myocardium (myo) at E11.5; and the expression is diminished by E14.5. mv/tv, mitral/tricuspid valve; ivs, interventricular septum. D, IHC showing Dnmt3b is predominantly expressed in AVC at E11.5. E, X‐gal staining showing the Nfatc1‐Cre mediated LacZ expression in the AVC at E11.5. F, RT‐qPCR showing that deletion of Dnmt3b resulted in increased Has2 expression at E11.5 and E14.5 (n=3). Error bars represent standard error. *Marks statistical significance (P<0.001, 2 factor ANOVA in [F]). ANOVA indicates analysis of variance; CKO, conditional knockout; IHC, immunohistochemistry; RT‐qPCR, quantitative real‐time polymerase chain reaction; WT, wild type.
Mentions: Previous knockout studies in mice have shown that Has2 is essential for development of cardiac valves and septa.51,56 Here, our DNA methylation analysis indicated that an ACGT site located within 1 kb of a previously determined enhancer of Has2, marked by enriched H3K27ac at E14.5,44 exhibited an increase in methylation as confirmed by Sequenom's MassArray (Figure 7A). Based on what is known about DNA methylation, we expected to see a decrease in Has2 expression at E14.5, with confirmation by qPCR analysis (Figure 7B). To further characterize its expression in the developing hearts, we carried out RNA in situ hybridization. The results showed that Has2 expression was predominately expressed in the endocardial cells and their mesenchymal progeny cells that form the primitive heart valves at E11.5, but its expression markedly diminished by E14.5 (Figure 7C), consistent with its function in endocardial to mesenchymal transformation around E11.5 for heart valve development.

Bottom Line: Quantitative real-time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2).Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer.Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co-expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY (A.A.C., M.L., A.A.M., Y.W., M.S., B.W., J.M.G., D.Z.).

Show MeSH
Related in: MedlinePlus