Abstract
Methylated non-CpGs (mCpH; H means A, C, and T) have emerged as key epigenetic marks in mammalian embryonic stem cells (ESCs) and neurons, regulating cell type-specific functions. In these two cell types, mCpHs show distinct motifs and correlations to transcription that could be a key in understanding the cell type-specific regulations. Thus, we attempted to uncover the underlying mechanism of the differences in ESCs and neurons by conducting a comprehensive analysis of public whole genome bisulfite sequencing data. Remarkably, there were cell type-specific mCpH patterns around methylated CpGs (mCpGs), resulted from preferential methylation at different contexts by DNA methyltransferase (DNMT) 3a and 3b. These DNMTs are differentially expressed in ESCs and brain tissues, resulting in distinct mCpH motifs in these two cell types. Furthermore, in ESCs, DNMT3b interacts with histone H3 tri-methylated at lysine 36 (H3K36me3), resulting in hyper-methylation at CpHs upon actively transcribed genes, including those involved in embryo development. Based on the results, we propose a model to explain the differential establishment of mCpHs in ESCs and neurons, providing insights into the mechanism underlying cell type-specific formation and function of mCpHs.
Highlights
In these cells, both methylated CpGs (mCpGs) and mCpHs are induced by DNA methyltransferase3a and 3b (DNMT3a and DNMT3b, respectively), with allosteric cooperation of DNMT3l9
Most of the CpGs were hyper-methylated (75–85%), the average methylation levels at CpHs were distinct across cell types. It was mostly abundant in neurons (>5%), abundant in embryonic stem cells (ESCs) derived from male (H1) and matured brain tissues (>1%), detectable in ESCs derived from female (H9), early-passage ESCs (HUES64), and immature brain tissues (0 to 5 year-old; about 1%), and mostly undetectable in other tissues (The heart, spleen, and lung;
The whole genome bisulfite sequencing (WGBS) samples were clearly clustered based on the tissue of origin by both CpG and CpH methylation patterns, indicating our integrated dataset well represents differential methylation pattern among cell types (Fig. 1b)
Summary
Both mCpGs and mCpHs are induced by DNA methyltransferase3a and 3b (DNMT3a and DNMT3b, respectively), with allosteric cooperation of DNMT3l9. Since these methyltransferases show much higher affinities at CpGs than CpHs, the mCpHs are spatially dependent on mCpGs3, 6, 10–13. Further analyses uncovered that DNMT3a and DNMT3b, differentially expressed in those two cell types, preferentially methylate cytosine in different contexts, resulting in distinct motifs and patterns of mCpHs in ESCs and neurons. The methylated CAGs were enriched in genes related to embryo development, indicating possible role of mCpHs on embryogenesis Based on these results, we suggest a differential CpH methylation model that could explain distinct features and functions of mCpHs in ESCs and neurons. Our results provide new insights on cell type-specific formation and function of mCpHs in mammals
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