Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

Michael J Ziller,Yingying Zhang,Christoph Bock,Hongcang Gu,Andreas Gnirke,Charles B Epstein,Alexander Meissner,Jing Liao,Thomas Lengauer,Patrick Boyle,Fabian Müller,Bradley E Bernstein,Dirk Schübeler

doi:10.1371/journal.pgen.1002389

Abstract

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.

Highlights

DNA methylation as a regulatory epigenetic mechanism is a widespread phenomenon [1]
We show that depletion of the de novo DNA methyltransferases results in a global reduction of non-CpG methylation levels
These findings further advance our understanding of cytosine methylation and describe its distribution among a large number of human cell types

Summary

Introduction

DNA methylation as a regulatory epigenetic mechanism is a widespread phenomenon [1]. In vertebrates, the CpG dinucleotide is the predominant target for methylation. In contrast plants exhibit frequent non-CpG (CpNpG and CpHpH) methylation and have established mechanisms to propagate CpNpG and asymmetric CpHpH methylation marks These differences between plants and mammals can partly be attributed to the presence of distinct methyltransferases. There are only three mammalian DNA methyltransferases exhibiting significant catalytic activity on DNA [6] These enzymes show a strong preference for CpG dinucleotides. Ectopic expression of murine Dnmt3a in D. melanogaster suggested that this enzyme is capable of de novo methylation that includes non-CpG targets [2] In line with these studies, it has been shown that mES cells exhibit detectable levels of non-CpG methylation and express Dnmt3a and Dntm3b at higher levels than most somatic cell types [2,7]. The involvement of DNMT1 in the establishment or maintenance of non-

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