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Published in final edited form as: Curr Opin Cell Biol. 2013 Mar 14;25(2):152–161. doi: 10.1016/j.ceb.2013.02.014

DNA methylation and methylcytosine oxidation in cell fate decisions

Kian Peng Koh 1,*, Anjana Rao 2,3,4
PMCID: PMC3649866  NIHMSID: NIHMS450110  PMID: 23498662

Abstract

Changes in cellular phenotypes and identities are fundamentally regulated by epigenetic mechanisms including DNA methylation, post-translational histone modifications and chromatin remodeling. Recent genome-wide profiles of the mammalian DNA “methylome” suggest that hotspots of dynamic DNA methylation changes during cell fate transitions occur at distal regulatory regions with low or intermediate CpG densities. These changes are most prevalent early during the course of cellular differentiation and can be locally influenced by binding of cell-type specific transcription factors. With the advent of next-generation quantitative base-resolution maps of 5-methylcytosine and its oxidized derivatives and better coverage of the genome, we expect to learn more about the true significance of these DNA modifications in the regulation of cell fate choices.

Introduction

In mammalian genomes, the major epigenetic modification of DNA is methylation at the 5-position of the cytosine base, often at symmetrical CG dinucleotides (CpG). DNA methylation is implicated in numerous cellular processes during development including genomic imprinting, X-chromosome inactivation and transposon silencing [1]. DNA methylation at promoters is often associated with inhibition of transcriptional activity, but more recent genome-wide profiling of the DNA “methylome” in plants and animals is revealing new biological functions of this mark in gene regulation [2,3]. In this review, we discuss recent genome-wide location profiles of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in different mammalian cell types, focusing on models of cellular differentiation from pluripotent or multipotent states towards restricted somatic cell lineages. This subject has been recently reviewed [4-6] but continues to evolve with increasing depth and coverage of whole genome sequencing efforts. Due to space limitations, we will not discuss the role of DNA methylation in the specification of germ cells and in cancer, for which we refer the reader to other recent reviews [7-9].

Dnmts and Tet proteins in stem cells and development

In somatic cells, DNA methylation is generally stable because the maintenance DNA methyltransferase Dnmt1 faithfully restores methyl marks on newly-replicated DNA strands. In contrast, dynamic genome-wide changes in DNA methylation occur during early embryogenesis, most notably in the paternal pronucleus of the zygote where replication-independent demethylation occurs shortly after fertilization, and during reprogramming of primordial germ cells [10]. Subsequently, methylation profiles in the genome are re-established by the de novo methyltransferases Dnmt3a and Dnmt3b as cells develop into restricted lineages. The tight regulation of DNA methylation and demethylation is crucial since Dnmt-deficient mouse embryos are impaired in development [11,12]. Similarly, Dnmt-deficient (and therefore hypomethylated) mouse embryonic stem (ES) cells can be maintained in culture but are impaired in differentiation [13,14].

The recently discovered Tet family of 2-oxoglutarate (2OG)- and Fe(II)-dependent 5-methylcytosine oxygenases [15,16] alter DNA methylation status by converting 5mC to 5hmC and the further oxidation products 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in DNA [17-19] (Figure 1). The loss of 5mC in the mouse paternal pronucleus occurs concomitantly with the appearance of Tet3-mediated hydroxymethylation [20-22••], a finding initially thought to represent a process of active and global DNA demethylation. The caveat is that both 5fC and 5caC are deaminated and read as T after bisulfite treatment and PCR amplification, and so cannot be distinguished from unmodified cytosine (C). Thus the apparent demethylation in the zygote could reflect oxidation of 5mC through 5hmC (which cannot be distinguished from 5mC by bisulfite sequencing [23]) to 5fC and 5caC followed by passive dilution of these oxidized products during cell cleavage [24], active base excision repair of 5fC and 5caC by thymine-DNA glycosylase (TDG) [17], or removal of the carboxyl group from 5caC by a putative decarboxylase [25] (Figure 1). A recent study suggests that the de novo Dnmts are also redox-dependent DNA 5-dehydroxymethylases in vitro, adding another potential route for active DNA demethylation [26].

Figure 1.

Figure 1

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The potential pathways of DNA demethylation mediated by TET proteins. In addition to the active pathways shown (see text for details), demethylation may occur passively through inhibition of maintenance methylation. Moreover, the methylcytosine oxidation products generated by TET proteins, like 5hmC, may function as stable marks that recruit chromatin “readers”,“writers’ or “erasers”.

Whereas targeted knockout of Tet3 in mice results in perinatal or embryonic lethality, Tet1-deficient mice can be born viable but runted, with evidence of strain-dependent and partially penetrant embryonic lethality [22••,27-29•]. In contrast, Tet2-deficient mice are viable and fertile (reviewed in [30]). In mouse ES cells, Tet1 and Tet2 are highly expressed and together sustain steady state 5hmC levels [31]. Like Dmnt-deficient ES cells, Tet-deficient ES cells can be sustained in self-renewing culture and exhibit enhanced trans-differentiation potential towards the extra-embryonic fate [27•,31]. However, ES cells in which Tet1 is depleted by siRNA are still able to differentiate into all embryonic germ-layer derivatives, but exhibit skewed disposition towards mesendoderm relative to neuroectoderm fates in vitro [31]. Whereas the precise role of Tet1 in regulating the differentiative potential of ES cells remains to be clarified, the impact of Tet2 loss-of-function in hematopoietic stem cell differentiation has been reported by several groups (reviewed in [30]). Collectively, these studies suggest that loss of Dnmt or Tet proteins can be dispensable for the maintenance of stem cell character but moderate differentiation phenotypes to varying extents as stem cells exit from pluripotent or multipotent states.

DNA methylation profiling in different cell states

Although promoter methylation contributes to repression of core pluripotency genes, such as Oct4 and Nanog, it is only a second-tier epigenetic change, occurring after histone H3(K9) methylation and heterochromatinization, to stably inhibit reactivation of these genes during differentiation [32] (Figure 2). Low CpG content promoters (LCP), which are generally associated with tissue-specific genes, tend to be constitutively highly methylated; on the other hand, high CpG content promoters (HCP), which often contain CpG “islands” (CGIs), remain unmethylated [33-35]. These findings imply that low concentrations of methylated cytosines at a promoter do not preclude gene activity; conversely, DNA demethylation is not sufficient for gene activation. Thus, a general view is that hypermethylation of HCPs, other than functioning as a secondary silencing mechanism for long-term stability and memory, is not a major mechanism of cell fate decision during development [35]. Nonetheless, a defined subset of CGIs exhibit de novo methylation during development. This occurs preferentially at intermediate CpG content promoters (ICPs), suggesting that weak CGIs are more prone to methylation during differentiation [33,34,36] (Figure 2).

Figure 2.

Figure 2

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Schematic representation of DNA methylation status surrounding a self-renewal gene (left) or a tissue lineage-specific gene (right) in pluripotent stem cells (A), in the lineage in which the gene is expressed (B) and in an alternative lineage is which the gene is silent (C). Arrows indicate the transcription start sites (TSSs). Raised circles denote CpG sites. Black filled circles are 5-methylcytosine, grey filled circles are hydroxymethylcytosine and white dots circles are unmethylated cytosine. Promoters depicted are high CpG-content promoters (HCP) (left) typical of pluripotency genes (as well as germline-specific and X-inactivated genes) and intermediate or weak CpG –island promoters (right). Most HCP of housekeeping genes and silent but “poised” developmental regulators (not shown) remain unmethylated regardless of transcriptional states. In contrast, weak CpG island promoters are preferential targets for de novo methylation in somatic cells[33]. Gene silencing may result from lack of activators or alternative repressive mechanisms independent of DNA methylation.

In recent years, focus has been diverted to intra- and inter-genic regions as potential sites of dynamic DNA methylation changes during development. Analysis of the human methylome in three normal tissue types representing each of the embryonic lineages – liver (endodermal), spleen (mesodermal) and brain (ectodermal) – revealed that most tissue-specific differentially methylated regions (DMRs) occur not in promoters, and also not in CpG islands, but in regions located within 2 kb of islands with comparatively low CpG densities, termed “CpG island shores” [37]. The functional role of these tissue-specific DMRs has been proposed to regulate alternative transcription during differentiation. Another group identified numerous “orphan” CGIs as conserved features in intragenic regions of the mammalian genome, where they often mark alternative promoters but are frequently subject to DNA methylation during development to suppress promoter functions [38]. Subsequently, several genome-scale DNA methylome studies with more comprehensive coverage revealed low promoter methylation and high gene-body methylation in highly expressed genes in both plants and animals [39-43]. The association of gene body methylation with increased transcriptional activity has been accounted for by some examples of tissue-specific intragenic CGI methylation functioning to repress alternative promoters [44•]. However, a negative correlation between cell type-specific intragenic CGI methylation and expression of associated genes has been observed in immune cell types [45•]. Alternatively, intragenic methylation marks may repel polycomb components and functionally antagonize polycomb-mediated repression [46•].

A recent genome-scale quantitative analysis of cytosine methylation in mouse ES cells and neuronal progenitors defines cell-type specific low-methylated regions (LMRs) with an average methylation of 30%. These are typically CpG poor regulatory regions distal to promoters and are occupied by DNA-binding factors[47••]. The study suggests that by locally influencing DNA methylation, binding of cell-type-specific transcription factors is necessary and sufficient to create LMRs. Consistent with a model of dynamic DNA methylation and demethylation at these regions, the LMRs also exhibit a strong pre sence of 5hmC and Tet1 binding [47••] (Figure 3). The new evidence of cell-type specific distal LMRs is in timely agreement with new data from the Encyclopedia of DNA Elements (ENCODE) project, a consortia effort to delineate all functional elements in the human genome[48]. In particular, the integrative analysis of genome-scale DNase I hypersensitive sites (DHSs), DNA methylation and transcription factor expression in diverse cell and tissue types reveals novel relationships between chromatin accessibility, DNA methylation and cell-type-specific transcription regulation [49•]. The project reports distal DHS to be largely cell selective and to coincide with transcription-factor binding sites that are, on average, less frequently methylated in cell types that express those transcription factors. The data strongly argue that cell-type specific DNA-binding regulators are key drivers of the accessibility landscape of the chromatin; a corollary is that cell-type-specific methylation patterning results from passive methylation of sites left vacant by transcription factors[49•].

Figure 3.

Figure 3

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Schematic representation of DNA methylation status surrounding a gene encoding a developmental regulator in stem cells (A), in a cell displaying tissue-specific expression and differentiation towards the correct lineage (B) and in a cell differentiating towards an alternative lineage (C). At silent but “poised” promoters of genes encoding developmental regulators (A), PRC 2 mediates tri-methylation of lysine 27 of histone 3 (H3K27me3) at the promoters to keep the gene silent but “poised” for activation upon initiation of specific developmental pathways. The low-methylated regions (LMR) distal to promoters (the LMR shown here is depicted as intergenic but it can also be found at intragenic sites) form dynamically during differentiation in a cell-lineage specific manner upon binding by transcription factors (TF) [47••]. Tissue-specific DMRs have been described at CpG island (CGI) shores [37]. High-density CGI promoters generally stay unmethylated regardless of lineage fate; appropriate silencing may involve histone modifications by PRC2. Within gene bodies, methylation levels have been positively correlated with gene expression. The gene body methylation reflects a global reduction in DNA methylation observed to occur during differentiation [42,43]. Alternative TSSs are shown as dashed arrows. Full gene activation may require suppression of these alternative TSSs, possibly by intragenic CGI methylation [44•].

Tracking DNA methylation dynamics during cellular differentiation

Early studies suggested that DNA methylation can function in a highly locus-specific manner, involving in some instances changes at a single cytosine, to regulate lineage-specific development (reviewed in [4,5]). However, these studies have been obscured by genome-scale data that, as described above, illustrate more often than not that promoter methylation and transcriptional activity do not follow each other in an obligatory fashion.

The tracking of DNA methylation changes at genome-wide scale during the time-line of cellular differentiation is the next approach to identify new differentially methylated regions (DMRs) critical in lineage specification. The best established model to date is the derivation of neuronal progenitors and terminally differentiated neurons from ES cells, for which multipotent progenitors and terminal neurons can be obtained with high efficiency. Using a murine system of in vitro differentiation from stem cells to neuronal progenitors to terminally differentiated neurons, a methylated DNA immunoprecipitation (MeDIP)-array analysis of promoter methylome demonstrated that weak CpG islands are preferentially controlled by DNA methylation during somatic differentiation [50], in agreement with the aforementioned studies. However, most of these changes already occur during the early stage of commitment to a multipotent progenitor state. A similar conclusion has been drawn from methylome maps obtained during the progressive differentiation of mouse hematopoietic stem cells into restricted myeloid or lymphoid lineages [45•,51•,52•].

The ultimate challenge is to understand the most dynamic DNA methylation changes occurring during the earliest phases of mammalian development in vivo. The reduced representation bisulfite sequencing (RRBS) approach has been used to produce base-resolution DNA methylomes in gametes and through implantation in the early pre-specified mouse embryo [53•,54]. These studies reveal that large scale changes in DNA methylation specifically occur during two transitions in development: (i) upon fertilization, as a gross reduction in methylation during the sperm-to-zygote transition predominantly at intergenic DMRs enriched for retroelements of specific families and (ii) during the transition between pre-implantation to early gastrulation stage, as massive remethylation from the ICM to the early-streak embryo[53•]. The latter transition involves a multitude of complex changes in early development as the primary germ layers are formed from the pluripotent epiblast. While the RRBS approach is currently the only one applicable to very small numbers of cells, a comprehensive genome-wide dissection of the epigenetic changes in the early germ-layer lineages is warranted.

Mapping 5-hydroxymethylcytosine – a new methylation variant in disguise

A major limitation of bisulfite-based approaches to methylation profiling is the inability to discriminate between 5mC and 5hmC [23]. Thus, all current state-of-the-art high resolution DNA methylome maps still require reinterpretation since what has been previously called methylation is really a sum of 5mC and 5hmC. For instance, the high gene body methylation levels of expressed genes may indicate enrichment of 5hmC at the expense of 5mC, as evident in terminally differentiated neural cell types[55•]. To add to the complexities, even less is known about 5fC and 5caC, which are more rare than 5hmC and are read as unmethylated cytosines in traditional bisulfite sequencing.

Following the discovery of 5hmC as a bona fide constituent of the mammalian genome, multiple groups have obtained genome-wide maps of 5hmC, especially in ES cells, using primarily antibody-capture or selective chemical labeling methods (reviewed in [56]). The consensus view from these studies is that 5hmC is located predominantly in gene bodies, enhancers and intermediate CpG-density promoters of both active and repressed genes in ES cells. Where 5hmC is found at transcription start sites (TSS) of inactive genes, repression is likely mediated by co-bound chromatin modifiers such as Sin3a or the Polycomb Repressive Complex (PRC) 2 [57••-59]. Recently, two groups successfully mapped 5hmC at base-resolution in ES cells using either selective chemical oxidation (oxBS-Seq) or Tet enzyme-assisted bisulfite sequencing (TAB-Seq)[60•,61•]. With a higher resolution and quantitative analysis not previously attained by affinity-based methods, TAB-Seq reveals 5hmC as most abundant at regions of low CpG content and predominantly in the CpG context. Almost half of the 5hmCs reside in distal regulatory elements, where 5mC and 5hmC often coexist at nearly equal levels at the same cytosine [61•], suggesting that active demethylation is strongest outside of genes. Supporting this model, a kinetic study using hydroxyMeDIP (hMeDIP) revealed dynamic association of 5hmC with transcription factor binding at distal regulatory sites of genes activated during differentiation, indicating that DNA hydroxymethylation is an early event of tissue-specific enhancer activation [62•].

The three Tet family genes exhibit marked differential expression patterns in different tissue types and cellular states and are likely recruited to different loci in the genome to orchestrate a diverse set of epigenetic landscapes (reviewed in [63]). The relevance of 5hmC in neurodevelopment has received attention since the base was also discovered at abundant levels in Purkinje neurons of the rodent brain [64]. Subsequently, 5hmC has been mapped as a stable and dynamically acquired mark at developmentally activated genes in neuronal cells during postnatal development through adulthood [55•,65•].

More recent examples of cell fate control by 5mC-5hmC conversion at lineage-specific genes have been attributed to Tet2: at the Hoxa cluster of developmental genes during retinoic acid-induced differentiation [66] and at gene promoters of specific myeloid genes during CEBPα-induced transdifferentation of pre-B cells into macrophages [67•]. Future studies are likely to illuminate more cases of Tet-dependent 5hmC marks at critical cell fate transitions, including the role of these marks in differentiated cells.

When normal tissues adapt to cell culture, global 5hmC content decreases rapidly whereas global methylation state increases over passaging, suggesting that culture conditions modulate the cellular DNA hydroxylation machinery and phenotype [35,68]. Thus, a full understanding of the complexities of 5mC and 5hmC function will require analyses with primary cells from human and mouse.

Conclusions and Perspectives

The recent advances in DNA methylome studies have rapidly changed previous perceptions of DNA methylation. We summarize with the following points (Figure 4):

  1. DNA methylation/hydroxymethylation changes at distal elements with low to intermediate CpG densities are likely more informative than changes at promoters/gene bodies in regulating cell fate.

  2. DNA methylation changes are most prevalent during the early stages of lineage commitment.

  3. Cell-type specific transcription factors can influence DNA methylation locally to cause tissue-specific expression, replacing the old dogma that DNA methylation blocks binding of transcription factors during differentiation.

  4. DNA methylation can be acquired during culture adaptation, so that what we know from in vitro cellular differentiation models may underestimate the changes occurring in vivo.

Figure 4.

Figure 4

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Schematic plot of the significance of 5mC/5hmC changes against a spatial axis of genome location and against a temporal axis of progressive lineage specification. The contour plot depicts a tall peak at distal regulatory regions and a lower peak at promoter and gene bodies along the spatial axis, indicating that the influence of cell type-specific dynamic changes in DNA methylation on lineage-specific gene expression is strongest at distal sites. These peaks align at an early time window along the temporal axis of increasing lineage commitment (or decreasing multilineage potency), indicating that most DNA methylation changes occur early when cells exit pluri- or multi-potency during the course of differentiation. In the “fourth” dimension, cell type specific transcription factors are shown above the DNA methylation contours to indicate that binding of transcription factors at distal regulatory elements influences DNA methylation status locally. The raised level indicated by the dotted lines denotes possible DNA methylation “artifacts” introduced by culture adaptation of cells and long-term passaging in vitro.

Collectively, the multiple efforts to profile 5mC and 5hmC in various cell states has generated a blueprint for further mechanistic understanding of these epigenetic marks in mammalian development. However, the variety of methods used, each with its strength and limitation, necessitates more fine-tuning of the landscape. With more advanced whole-genome sequencing studies unraveling regions of low CpG densities as hot spots of dynamic DNA changes critical in cell fate decisions, our current understanding of this fundamental epigenetic process may still represent only the tip of the iceberg. In the near future, the ideal approach is to obtain unbiased genome-scale base-resolution maps of 5mC and 5hmC (and even 5fC and 5caC) to venture beyond the CpG islands and shores to discover novel DMRs during differentiation.

Despite the allure of genome-wide approaches, the utility of candidate gene loci-specific studies should nonetheless remain relevant. As shown in the case of Tet2-regulated myeloid target genes, the critical switch in lineage-specific gene expression is accompanied by fairly subtle changes in DNA methylation [67•]. Thus, the dynamic range of DNA methylation changes during lineage specification appears narrow, yet may exert a threshold effect sufficient to trigger more widespread chromatin remodeling to affect key cell fate decisions. In this regard, candidate loci-specific probing of DNA methylation changes can be more powerful than genome-wide overviews to reveal the subtle effects.

Beyond mapping these epigenetic marks, the ultimate goal is to harness these insights to devise epigenetic-based strategies to skew in vitro differentiation of stem cells into desired cell types for multiple applications in drug testing, disease modeling and cellular replacement therapies. This may require precise definition of the cause-and-effect relationship of loci-specific changes (or DMRs) and cellular phenotype, perhaps the most elusive question in the field of epigenetics. Unlike single gene studies in which loss-of and gain-of function approaches are available to confer precise functions, histone and DNA modifications are not readily amenable to precise targeting. In view of the tight association between DNA binding of lineage-specific transcription factors and methylation states of regulatory elements, one can postulate testing such hypothesis using fusion constructs of sequence-specific binding factors and chromatin-modifying catalytic domains to enhance skewing towards specific lineages. The revolutionary reprogramming of somatic cells to induced pluripotent stem cells as well as directed the interconversion between lineages by forced introduction of key transcription factors already demonstrate that cellular identity is inherently more plastic than previously thought. Future endeavor promises to define a limited set of general rules and key factors sufficient to modulate the epigenome to attain the correct cell fate.

Highlights.

  • DNA methylation changes at distal elements of low CpG densities regulate cell fate.

  • These changes are most prevalent during the early stages of lineage commitment.

  • Cell-type specific transcription factors locally influence DNA methylation.

Acknowledgements

We apologize to those whose work could not be cited due to space limitations. Research in the laboratory of Kian Koh is supported by the Fonds voor Wetenschappelijk Onderzoek Research Foundation – Flanders (G.0C56.13N and G.0632.13), the Ministerie van de Vlaamse Gemeenschap and the Marie Curie Career Integration Grant (PCIG-GA-2012-321658). Research in the lab of Anjana Rao is supported by NIH R01 grants HD065812, AI44432 and CA151535, grant RM1-01729 from the California Institute for Regenerative Medicine, and Translational Research Program Award 6187-12 from the Leukemia Society of America.

Footnotes

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