DNA methylation is developmentally regulated for genes essential for cardiogenesis.
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.
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
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Mentions: Next, we chose up to 14 ACGT sites with a range of different tag counts, representing 0%, 25%, 50%, 75%, or 100% methylation by massively parallel sequencing, and determined their levels of methylation by MassArray. The results indicated that tag count was inversely correlated with the percentage of cytosine methylation (Figure 3A), thereby confirming the precision of the MSFE/MPS in quantifying methylation level, ie, tag counts measured accurately both the global and regional DNA methylation. We then set out to investigate how much methylation changed in the developing hearts between E11.5 and E14.5. After normalization by sequencing depths, ACGT sites with at least 1 sequencing tag in any of the 4 samples were evaluated for differential methylation using 2 complementary approaches. We used EdgeR, which modeled the tag counts by a negative binomial distribution, to determine ACGT sites that showed differential methylation. The result indicated that the majority of the ACGT sites were not differentially methylated in the developing hearts between the 2 stages, as <1% of sites were found to have different tags (nominal P value <0.05) (Figure 3B). Among the small fraction (2901) of the ≈1.64 million analyzed ACGT sites that were differentially methylated, 1946 (67.1%) and 955 (32.9%) sites exhibited increased and decreased methylation in the late stage hearts, respectively (FDR<0.05) (Figure 3C).38 Of note, for the majority of these sites, the degree of difference was <50%, with no sites switching from a fully methylated to an unmethylated state.
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.).