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. 2015 Jul 4;168(4):1219–1225. doi: 10.1104/pp.15.00543

DNA Methylation within Transcribed Regions

Taiko K To 1,2,1, Hidetoshi Saze 1,2,1, Tetsuji Kakutani 1,2,*
PMCID: PMC4528756  PMID: 26143255

Although DNA methylation within transcribed genes is commonly found in diverse animals and plants, recent advances highlight the remaining mystery regarding intragenic DNA methylation.

Abstract

DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation.

In plant genomes, DNA methylation is found not only in promoters but also within transcribed regions (Fig. 1; Table I). The characteristics of DNA methylation within transcribed regions differ from those in promoters. For promoters, high levels of DNA methylation are found only in silent genes and transposable elements (TEs). In contrast, actively transcribed genes do not have much DNA methylation around their promoter regions. However, in regions sufficiently downstream (approximately 500 bp) from the transcription start sites (TSSs), substantial amounts of DNA methylation are often found even in transcribed genes (Zhang et al., 2006; Zilberman et al., 2007; Fig. 1). DNA methylation within transcribed genes is commonly found in plants and animals. Furthermore, intragenic DNA methylation has unique features that are evolutionarily conserved among these organisms (Zemach et al., 2010), implicating one or more basic functions. However, the control and biological role of intragenic methylation still remain largely unknown. Here, we provide an overview of recent findings about intragenic DNA methylation and discuss remaining questions.

Figure 1.

Figure 1.

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Schematic diagram of intragenic DNA methylation in Arabidopsis (Arabidopsis thaliana). A, DNA methylation at TSSs and/or in promoters. Intensive CpG methylation (red bars) and non-CpG methylation (blue bars) are found within the TSSs and promoters of silent genes and TEs. Their transcription is repressed by the methylation. B, DNA methylation in intron. TEs or repeats located in the introns of transcribed genes are methylated in both CpG and non-CpG contexts. When such DNA methylation is lost, immature transcripts are detected, probably because of the cryptic poly(A) addition signal within the repeats. C, DNA methylation in gene bodies. Only CpG methylation is found in the gene bodies of actively transcribed genes. Both exons and introns are methylated except for TSS-proximal regions.

Table I. DNA methylation in the promoter and transcribed region.

Methylation in Promoter Methylation in Transcribed Region
Intragenic Heterochromatin Gene Body Methylation
Transcription Silent Any type Housekeeping
Contexts Both CpG and non-CpG Both CpG and non-CpG CpG
Exon/intron Mainly in intron Both exon and intron
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We review two types of intragenic methylation, which differ in relation to transcription activity, exon/intron distribution, and the context of methylated cytosine (Table I). In plant genomes, cytosine can be methylated in both CpG and non-CpG contexts. Non-CpG methylation is associated with methylation of histone H3 Lys 9 (H3K9me), which is an epigenetic mark of silent chromatin conserved among eukaryotes (Johnson et al., 2007; Inagaki et al., 2010). Methylation is found in both CpG and non-CpG contexts in promoters of silent genes and TEs. The first type of intragenic methylation we discuss is found mainly within intron, and both CpG and non-CpG contexts can be methylated. Therefore, this type of DNA methylation can be understood as islands of silent chromatin found in introns of active genes (Fig. 1).

The other type of intragenic methylation, called gene body methylation, is found primarily in exons but also in introns (Chodavarapu et al., 2010), and almost always only CpG sites are methylated. Very interestingly, this type of intragenic methylation is found in constitutively transcribed genes. Inducible genes and developmentally regulated genes generally do not have CpG gene body methylation (Aceituno et al., 2008; Coleman-Derr and Zilberman 2012). In this review, we discuss these two types of intragenic DNA methylation separately, dealing first with intronic heterochromatin and subsequently with gene body methylation in constitutively transcribed genes.

CONTROL OF INTRAGENIC HETEROCHROMATIN

Substantial numbers of intragenic heterochromatin islands are found for both animal and plant genes, especially within the introns (van de Lagemaat et al., 2006; Sela et al., 2007; Nystedt et al., 2013; Seymour et al., 2014; West et al., 2014). Most of these examples of intronic heterochromatin reflect insertions of TEs. In the human genome, 60% of TEs are localized within introns that comprise only 24% of the genome; therefore, TEs are much enriched in introns. Similarly, most plant genomes have many TEs in introns. One exception is the genome of Arabidopsis, which contains relatively few intronic TEs. A recent analysis estimated that only 0.7% of annotated genes in the Arabidopsis genome contain intronic TEs (Le et al., 2015). However, genome organization of Arabidopsis is exceptional; most plant species have a large number of TEs within introns. A recent maize (Zea mays) epigenome study showed that approximately 10% of genes contain intronic TEs >1 kb in length (West et al., 2014). The introns of Arabidopsis lyrata, which diverged from Arabidopsis around 10 million years ago, contain many more TEs than those of Arabidopsis, reflecting the lineage-specific expansion/contraction of TE sequence within genic regions (Seymour et al., 2014). The genome of Norway spruce (Picea abies), the first genome available for gymnosperm, is 100 times larger than Arabidopsis, but the number of genes (approximately 28,000 genes) and exon sizes are similar (Nystedt et al., 2013). In contrast, the P. abies genome contains many genes harboring long introns, mainly reflecting insertion of long-terminal-repeat-type retrotransposons. These findings suggest that genomes are relatively tolerant of the presence of TEs, especially in intronic sequences, and intronic TEs are widespread in the plant genomes.

Many of these genes with intronic TEs are actively transcribed. Although the presence of TEs negatively correlates with transcription of nearby genes for a genome-wide trend (Hollister and Gaut, 2009; Wang et al., 2013b), a reasonably high level of expression is often found in the TE-bearing genes, comparable with that of genes without TE insertion (Nystedt et al., 2013; West et al., 2014). Importantly, TEs within introns often have repressive epigenetic marks, such as DNA methylation and H3K9me (Fig. 2). That is the case for intronic TEs in both maize and A. lyrata. Even in the exceptionally compact genome Arabidopsis, long introns often contain non-CpG methylation, mainly associated with TE insertions there (Saze et al., 2013; Le et al., 2015; Fig. 1). The level of non-CpG methylation in long introns in Arabidopsis is comparable with that in rice (Oryza sativa), although the number of such heterochromatic introns is much higher in rice (Fig. 3), as in other plant species. The intronic TEs are sometimes indistinguishable from copies in intergenic regions in terms of silent marks, such as non-CpG methylation, H3K9me, and small RNAs (Fig. 2).

Figure 2.

Figure 2.

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An intronic TE associated with heterochromatic epigenetic marks within the actively transcribed gene AT3G05410 in Arabidopsis. Top to bottom tracks: RNA-sequencing reads (green) in wild-type Columbia (Col; Saze et al., 2013), DNA methylation at CpG (blue), CHG (light blue), CHH sites (pink; H. Saze, unpublished) in wild-type Col, chromatin immunoprecipitation-Chip hybridization signals of H3K9me2 in wild-type Col (blue; Inagaki et al., 2010), small RNAs (magenta; Lister et al., 2008), and Arabidopsis gene model (The Arabidopsis Information Resource 10 [https://www.arabidopsis.org]). Non-long-terminal-repeat retrotransposon ATLINE2 is inserted in the antisense orientation relative to AT3G05410.

Figure 3.

Figure 3.

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Heterochromatic introns in the genomes of Arabidopsis and rice. The figure is adapted from Saze et al. (2013). A, Proportion of methylated cytosine at CHG sites compared among introns of different length. The value was derived from the sum of mapped cytosines in each class. Long introns tend to have more CHG methylation in both Arabidopsis and rice. B, The rice genome contains many more long heterochromatic introns than the Arabidopsis genome. Introns longer than the seventh intron of IBM1 (>1,975 bp) are plotted with the proportion of methylated cytosine in CHG sites, a hallmark of heterochromatin. Red and blue dots represent introns of Arabidopsis and rice, respectively.

Genes containing islands of heterochromatin within their introns are generally transcribed properly. An interesting question is what mechanisms allow plants to mask the deleterious effects of intronic TE sequences associated with repressive epigenetic marks. Indeed, recent studies identified factors in plants required for proper transcription of genes containing repressive epigenetic marks in intronic regions (Saze et al., 2013; Wang et al., 2013a; Coustham et al., 2014; Lei et al., 2014). One of the factors, ENHANCED DOWNY MILDEW2 (EDM2), was initially identified as a factor required for plant resistance to pathogen and was subsequently found to be required for proper transcription of a disease resistance gene, Resistance to Peronospora parasitica7 (RPP7), that contains a number of intronic TEs forming heterochromatic domains (Tsuchiya and Eulgem, 2013). EDM2 has Plant Homeodomain domains that recognize H3K9me and a putative RNA methyltransferase domain in its C-terminal part (Lei et al., 2014; Tsuchiya and Eulgem, 2014). Another factor, INCREASE IN BONSAI MATHYLATION2 (IBM2)/ANTI-SILENCING1 (ASI1)/SHOOT GROWTH1 (SG1), was identified as a gene responsible for DNA hypermethylation of gene bodies (Saze et al., 2013), an antisilencing effect for a transgene (Wang et al., 2013a), and pleiotropic developmental abnormalities (Coustham et al., 2014) in these Arabidopsis mutants. IBM2 contains the bromo-adjacent homology domain that likely binds to chromatin and an RNA recognition motif, although the direct target of IBM2 is still unclear. The mutant phenotypes are partly due to a transcription defect at the histone H3K9 demethylase gene IBM1, the seventh intron of which contains a repetitive sequence, which is similar to organellar genomes and modified with CpG and non-CpG DNA methylation (Rigal et al., 2012). The ibm1 mutation is known to cause a genome-wide genic DNA hypermethylation in non-CpG sites, accompanied by pleiotropic developmental defects (Saze et al., 2008). Indeed, the IBM1 transgene without the sequence of heterochromatic domain in the intron can rescue these ibm1-like phenotypes of ibm2. In edm2 and ibm2 mutants, in addition to the IBM1 gene, the full-length transcript of genes containing heterochromatic TE was reduced, and instead transcripts were prematurely terminated and polyadenylated within the associated TE sequences (Saze et al., 2013; Tsuchiya and Eulgem, 2013). Interestingly, RNA polymerase II elongation over the heterochromatic domains within introns is not affected in ibm2, suggesting that IBM2 is not required for passage of PolII; it more likely affects posttranscriptional processes, such as efficient splicing of heterochromatic introns and/or suppression of cryptic poly(A) signal sequences in the intronic repeats. Although EDM2 and IBM2 preferentially localize to the intronic heterochromatin, it is currently unclear how the maintenance of repressive heterochromatic states within the actively transcribed region is achieved.

More counterintuitively, maintenance of heterochromatin marks such as DNA methylation and H3K9me within introns can be important for proper expression of genes containing that, as loss of heterochromatic modifications in mutants such as decrease in DNA methylation1 and methyltransferase1 (met1) leads to a transcription defect of these genes (Le et al., 2015). For example, a reduction of DNA methylation of the repetitive element in the IBM1 gene in the met1 mutant results in a premature termination of the transcripts in the upstream of the repeat sequence (Rigal et al., 2012). In addition, a reduction of H3K9me at intronic TEs in the triple mutant of H3K9 methylase genes suvh4-suvh5-suvh6 affects transcription of the RPP7 gene (Tsuchiya and Eulgem, 2013). A relationship between non-CpG methylation in the intronic region and transcription also occurs in maize, where non-CpG methylation at exon-intron junctions inhibits alternative splicing (Regulski et al., 2013). In winter wheat (Triticum aestivum), cold treatment for induction of vernalization induces non-CpG methylation at TEs present in the intron of the VARNALIZATION-A1 gene, which is associated with the transcriptional activation of the gene (Khan et al., 2013). Thus, repressive epigenetic modifications on repeats and TEs in intronic regions might have a role in regulation of gene activation, beyond the silencing of TEs. Indeed, the intronic repeat in the IBM1 gene could function as a sensor for a fine-tuning mechanism for global changes in DNA methylation, where a reduction of DNA methylation at the repeat can reduce IBM1 transcripts, which eventually induce a genome-wide DNA hypermethylation (Rigal et al., 2012). The IBM1 intronic repeat emerged before the speciation of Arabidopsis, suggesting that an acquisition of feedback regulatory mechanisms for DNA methylation might be beneficial (Rigal et al., 2012; Saze et al., 2013). On the other hand, intronic TEs often show intraspecies insertion/deletion polymorphisms (Liu et al., 2004; Ziolkowski et al., 2009), suggesting that the modification of intronic TEs with repressive epigenetic marks might be a short-term adaptation mechanism to neutralize the deleterious effects of TE insertion.

GENE BODY METHYLATION

We have discussed intronic DNA methylation at both CpG and non-CpG sites, a signature of heterochromatin. The other type of intragenic DNA methylation is gene body methylation, which is found in euchromatic regions and is predominant in CpG sites (Lister et al., 2008; Feng et al., 2010; Zemach et al., 2010; Regulski et al., 2013). Gene body methylation of transcriptionally active genes is a common feature in diverse eukaryotes, suggesting a conserved function(s) (Feng et al., 2010; Zemach et al., 2010). Gene body methylation level positively correlates with gene expression level (Lister et al., 2008; Feng et al., 2010; Zemach et al., 2010). The connection of gene body methylation to transcription level can also be seen in the allelic pair of genes in X chromosomes of female mammals; genes on the active X chromosome have higher levels of gene body methylation than their counterparts on the inactive X chromosome, despite their nearly identical sequences (Hellman and Chess, 2007). Interestingly, the correlation between gene body methylation and transcription is more clearly observed in proliferating cells than in nonproliferating cells, suggesting that gene body methylation might be connected to cell proliferation in mammals (Aran et al., 2011). In plants, although TEs and silent genes have cytosine methylation in both CpG and non-CpG contexts, gene body methylation is found only in the CpG context (Table I). Gene body methylation is maintained by the maintenance methylase DNA METHYLTRANSFERASE1 (MET1) and its cofactor proteins in the VARIANT IN METHYLATION family (VIM1–VIM3) in Arabidopsis. Both classes of proteins are conserved in mammals; MET1 and VIMs are Arabidopsis orthologs of DNA Methyltransferase1 and Ubiquitin-Like with Plant Homeodomain and Ring Finger Domains1 in vertebrates, respectively (Finnegan and Kovac, 2000; Woo et al., 2008).

In regard to gene body methylation, two important questions remain unsolved: what is the biological role(s) of gene body methylation, and how is the gene body methylation pattern generated?

To address the second question, one approach is to identify mutants affecting gene body methylation. When DNA methylation is examined genome wide in 86 mutants of Arabidopsis known to have DNA methylation defects, none of the mutants other than met1 or vim1 vim2 vim3 triple mutants abolished gene body methylation (Stroud et al., 2013). Histone modifications and non-CpG methylation in heterochromatin are controlled by multiple pathways (Matzke and Mosher, 2014), but CpG methylation in gene bodies seems to be controlled by a simpler manner, by the maintenance methylation machinery. This simplicity may make sense, because CpG methylation in gene bodies tends to be maintained very stably and can even be inherited over generations (Vongs et al., 1993; Kakutani et al., 1999; Kankel et al., 2003; Saze et al., 2003). Nonetheless, it would be interesting to know how the pattern of gene body methylation is generated initially, before it is maintained transgenerationally.

Recently, several new results have been published with regard to the first question: the biological role of gene body methylation. Takuno and Gaut (2012, 2013) addressed the evolutional role of gene body methylation. Comparison of genomic bisulfite sequencing data with the phenotypic database showed that phenotypic effects could be seen in 55.7% of body-methylated genes, whereas such effects were only associated with 26.2% of the undermethylated genes, indicating that body-methylated genes are functionally more important. In addition, body-methylated genes show significantly lower nonsynonymous to synonymous substitution rates, which is consistent with the phenotypic results; amino acid sequences of functionally important genes should be conserved. Interestingly, both nonsynonymous and synonymous substitution rates are lower in body-methylated genes. This was especially surprising considering that methylcytosine is expected to be mutagenic and increase C/G-to-T/A mutation rates through spontaneous deamination (Bird, 1980; Pfeifer, 2006). The low mutation rate may be due to a low frequency of CpG sites, or due to low nucleosome occupancy, which may allow easy access of repair machinery (Meier and Thoma, 2005; Ataian and Krebs, 2006), although, according to the calculation by Takuno and Gaut (2012), each of these factors alone does not fully explain the low mutation rate of body-methylated genes.

Body-methylated genes also evolve slower in mammals and invertebrates. The comparative analysis of human DNA methylome data with human-macaque and human-mouse protein evolutionary rates revealed that gene body methylation is negatively correlated with protein evolutionary rate, whereas promoter methylation correlates positively (Chuang and Chiang, 2014). Similarly, genes with heavy body methylation are evolutionarily conserved and enriched for housekeeping functions in invertebrates (Sarda et al., 2012).

In mammals and other animals, accumulating evidence suggests a link between gene body methylation and alternative splicing. Depletion of DNA methylation by the inhibitor 5-aza-dC causes the failure of methyl-CpG-binding protein2 recruitment to the specific exons, resulting in exon skipping of the alternatively spliced sites (Maunakea et al., 2013). DNA methylation can also control alternative splicing by modulating binding of CCCTC-binding factor and RNA polymerase II pausing (Shukla et al., 2011). Interestingly, artificially established repressive marks driven by small interfering RNAs can cause an increase in DNA methylation and ectopic inclusion of noncanonical exons (Schor et al., 2013). A role of gene body methylation in alternative splicing is also observed during caste differentiation of social insects (Lyko et al., 2010; Bonasio et al., 2012; Terrapon et al., 2014). It would be interesting to know how certain methylated exons, but not other methylated exons nearby, can be specifically recognized by these factors.

The role of gene body methylation may be related to the interaction of the host with TEs. One possible role could be to shelter genes from TE insertion. Maize transposon Robertson’s Mutator (Mu) inserts preferentially within genes. Genome-wide mapping of insertion sites of Mu in the maize genome revealed that it preferentially inserts within unmethylated regions (Liu et al., 2009; Regulski et al., 2013). It would be interesting to examine if this can be applied to insertion sites of TEs other than Mu. Another possible role of gene body methylation could be a trigger to silence TEs but not genes, generating their differential methylation. If the host methylates the body of transcribed sequences, this may affect transcription of TEs more than that of genes. The small difference could be amplified by positive feedbacks of active and inactive states (see Inagaki and Kakutani, 2012 for a detailed discussion).

An interesting feature of gene body methylation is that it is found in housekeeping genes (Aceituno et al., 2008; Sarda et al., 2012). In other words, genes responding to environmental or developmental signals generally do not have body methylation. The link between gene body methylation and responsiveness has been suggested recently. In diverse eukaryotes including mammals, fish, and plants, DNA methylation anticorrelates with a histone variant H2A.Z (Zilberman et al., 2008; Conerly et al., 2010; Zemach et al., 2010; Valdés-Mora et al., 2012). H2A.Z is a histone variant evolutionarily conserved from yeast (Saccharomyces cerevisiae) to higher eukaryotes (Raisner and Madhani, 2006). It is predominantly localized near TSSs and is required for the poised state of transcription initiation, reducing nucleosome density and increasing DNA accessibility around TSSs (Fan et al., 2002; Guillemette et al., 2005; Tirosh and Barkai, 2008; Hu et al., 2013). These antagonistic epigenetic marks at TSSs may decide whether the gene is silenced (methylated) or kept poised for activation (H2A.Z). In Arabidopsis, the anticorrelation of H2A.Z against DNA methylation is also observed within gene bodies (Zilberman et al., 2008; Coleman-Derr and Zilberman, 2012). The genes containing H2A.Z over gene bodies are enriched in genes responsive to environmental or developmental stimuli (Coleman-Derr and Zilberman, 2012). Conversely, gene body methylation is found predominantly at constitutively expressed housekeeping genes and showed clear negative correlation against gene responsiveness (Aceituno et al., 2008). This global anticorrelation may imply that DNA methylation and H2A.Z exclude each other. Actually, the global loss of CpG methylation in Arabidopsis met1 causes an increase in H2A.Z occupancies in regions normally methylated in the wild type (Zilberman et al., 2008). On the other hand, disruption of the H2A.Z gene rarely alters the levels and patterns of gene body methylation, suggesting that H2A.Z does not exclude DNA methylation (Coleman-Derr and Zilberman, 2012). Therefore, gene body methylation might function upstream by preventing the incorporation of H2A.Z within gene bodies of housekeeping genes. It will be interesting to determine if the gene body methylation affects transcription stability/instability.

CONCLUSION AND PERSPECTIVE

Here, we discussed two topics: (1) mechanisms to handle intronic heterochromatin, and (2) gene body methylation of housekeeping genes.

Mechanisms to control intronic heterochromatin were discussed first. Although the Arabidopsis genome does not have much heterochromatin in introns, genomes of typical plant species have a large amount of intronic heterochromatin. The mechanism to mask the deleterious effect of heterochromatin in intron by EDM2 and IBM2/ASI1/SG1 should be important for evolution. An interesting possibility is that the intronic heterochromatin could also be a source for unique gene controls. For example, the intronic repeat in the IBM1 gene could function as a sensor for a fine-tuning mechanism for global changes in DNA methylation (Rigal et al., 2012). It might be interesting to search conserved intronic heterochromatin in various species with a potential regulatory role of gene expression through its epigenetic changes.

We also discussed control and biological role of gene body methylation. In plants, gene body methylation pattern, which is prevalently found in CpG sites, should be established transgenerationally. For the transgenerational establishment of gene body methylation, possible effects of histone variants and histone modifications as well as environmental and genetic variations could be an exciting research area for future exploration. A big question would be possible biological roles of gene body methylation. Despite conservation during evolution, the roles still remain mysterious.

Acknowledgments

We thank Eric Richards and Shohei Takuno for comments on the article.

Glossary

TE

transposable element

TSS

transcription start site

Mu

Robertson’s Mutator

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