Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells

Abstract

DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.

Non-CG Methylation in the Human Genome

“…The genome-wide distribution of mCH is neither uniform nor random but is greatly enriched in several genomic features, including gene bodies (31, 32, 105), repeat elements (3, 34, 113), and inactive enhancers (60), and it tends to be absent in active enhancers (56), promoters (31, 48, 56, 105), transcription factor binding sites (56), and mCH deserts, which are megabase-scale regions that have been recently identified in brain (54) (Figure 3). In addition, mCH preferentially occurs in regions of low CG dinucleotide density (32).…”

“…Another interesting observation in ESCs is that gene body mCH does not occur evenly in both strands; the antisense strand is non-CG hypermethylated (56). A recent study showed that this phenomenon derives from the skewed frequency of methylation-prone motifs (5′ACA3′) on the two strands (34). The functional consequences of this strand-specific mCH in the gene body remain unclear.…”

“…Although mCH is present in various cell and tissue types, evidence for its potential function is just beginning to emerge (4, 5, 15, 27, 32, 34, 57, 61, 71), and its true biological role is still unclear and controversial (3, 23, 63, 71, 86, 90). The DNA methylation profile of human ESCs revealed a strong correlation between mCH and adjacent mCG, suggesting that the presence of mCH may be the result of hyperactivity of nonspecific de novo methylation that is simply a tolerated by-product of mCG pathways (90, 113).…”

Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge

“…Many genes do not contain identifiable CpG-enriched regions that are methylated within their regulatory regions (Bird, 2002). However, the methylation also occurs in non-CpG regions in mammalian embryonic stem cells (Ramsahoye et al, 2000;Guo et al, 2013). The resulting methyl-group lies within the major groove of the DNA double-helix (Vargason et al, 2000) and there it can act as a docking site for binding proteins.…”

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants

“…However, although DNA methylation occurs mostly on CpG dinucleotide sites in mammals and human (Bird 2002), it can also found in other context including CHG and CHH (where H is A, C or T), notably in mammalian embryonic stem cells Laurent et al 2010;Lister et al 2009), human pluripotent cells (Guo et al 2014;Ziller et al 2011), oocytes (Kobayashi et al 2012;Tomizawa et al 2011), mammalian brain cells (Lister et al 2013;Xie et al 2012) and plant genomes (Becker et al 2011;Feng et al 2010;Zemach et al 2010;Zhong et al 2013). Whole genome bisulfite sequencing experiments using second-generation sequencing based on the Illumina/Solexa sequencing-by-synthesis technology produced the first single base resolution methylome in Arabidopsis thaliana.…”