Abstract
Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.
Highlights
DNA methylation is an epigenetic modification that plays an important role in many biological processes like genomic imprinting, X chromosome inactivation, embryogenesis, cellular differentiation, and transposon silencing [1]
DNA methylation is established by the de novo DNA methyltransferases DNMT3A and DNMT3B [2,31]
Previous studies have demonstrated the activity of the LSD1/Mi2/NuRD complex on the enhancers of pluripotency genes (PpGs) [29]
Summary
DNA methylation is an epigenetic modification that plays an important role in many biological processes like genomic imprinting, X chromosome inactivation, embryogenesis, cellular differentiation, and transposon silencing [1]. The DNA methyltransferases (DNMTs) DNMT3A and DNMT3B establish DNA methylation during early embryogenesis [2] and the post-replication maintenance of DNA methylation is largely performed by the enzyme DNMT1 [3,4]. DNA methylation involves the transfer of a methyl group from S-adenosyl methionine to the cytosine base mostly in CpG dinucleotides of the DNA [1,5]. The CpG dinucleotide is underrepresented in the mammalian genome [6,7] except in regions distinctly identified as CpG islands, which tend to be largely unmethylated [8]. CpG islands are found overlapping with the promoters of constitutively expressed house-keeping genes and are associated with regulatory regions of imprinted genes. The promoters of tissue-specific genes and distal regulatory elements, such as enhancers, usually constitute
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