DOI: 10.1111/eci.12403

REVIEW ESCI award lecture: regulation, function and biomarker potential of DNA methylation € beler*,† Dirk Schu *

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, University of Basel, Faculty of Science, Basel, Switzerland

ABSTRACT Methylation of DNA and modifications of histones have emerged as intricately involved in gene regulation as they cross-talk and respond in multiple ways to the activity of transcription factors. Measuring these epigenome components has become a powerful tool to identify regulatory principles and biomarkers that predict cellular state during development or disease. Here, I will focus on DNA methylation as a reversible epigenetic modification of DNA that has been studied in great detail at the level of the genome. Recent advances in sequencing have identified unexpected dynamics of this modification, which are tightly linked to gene regulation. Understanding how DNA methylation patterns are read and how they contribute to regulation will be critical to interpret and utilize genomic maps of DNA methylation. As these patterns are dynamic during cellular differentiation and perturbed in disease, they present an opportunity to use DNA methylation as a biomarker. Keywords biomarker, DNA methylation, epigenome, functional genomics, gene regulation. Eur J Clin Invest 2015; 45 (3): 288–293

Gene regulation Precise activation of genes is a key regulatory process in biology. It enables cell specification in metazoans and its misregulation has been linked to many diseases. Genomic DNA sequence remains largely invariant across different mammalian cell types, while their expression patterns vary widely reflecting their distinct functions. The correct spatio-temporal control of gene activity is primarily encoded within regulatory elements such as enhancers and promoters, which are interpreted by DNA-binding proteins such as transcription factors. These factors act within a chromatinized template consisting mainly of nucleosomes. Chromatin can be changed in multiple ways through post-translational modification of histones or their positioning. Moreover, DNA itself can be modified through addition of a methyl group to the base cytosine. These changes in chromatin structure can define DNA access for transcription factors [1] and furthermore enable temporal integration of regulatory events through dynamic processes including cell division and organism development. These modifications of DNA and nucleosomes are referred to as ‘epigenetic’ as they provide a potential means to dynamically and reversibly change the context of DNA sequence in a heritable fashion. It has been correctly criticized that the term ‘epigenetics’ is used too loosely in chromatin research and referred to any biology that involves chromatin and DNA methylation [2]. Indeed, in most cases, we know little how much of the observed


complexity in histone and DNA modifications is instructive for gene regulation or reflective of gene activity and how observed patterns are inherited. A more parsimonious definition for epigenetics has thus been put forward, which leaves the question of regulatory activity and inheritance open [3]. These discussions are not merely semantic but critical as they highlight the need to better understand the mechanisms of regulation and they remind us to not get carried away by intriguing correlations or the beauty of apparently simple models. In this review, on the occasion of the ESCI 2014 award for Excellence in Basic/Translational Research, I will focus on DNA methylation as a mammalian epigenetic mark that has been analysed in much detail and with a particular potential as a biomarker due to its accessibility for detection.

DNA methylation Methylation of cytosines is a reversible modification of DNA. It is referred to as epigenetic as it does not interfere with base pairing. In eukaryotes, it occurs mostly at cytosines next to guanine. The resulting CG sequence symmetry is used to propagate methylation through cell division as the hemimethylated state that occurs after the replication fork is recognized by the maintenance methyltransferase DNMT1 and assisting proteins [4–6]. Thus, once established by the de novo DNA methyltransferases DNMT3A and DNMT3B [5,7], this maintenance mechanism enables propagation through cell division

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until removed by an active or passive process [8]. The existence of this inheritance mechanism, first postulated almost 40 years ago [9,10], makes DNA methylation particularly attractive as an epigenetic means to memorize information over time. To fulfil this function, DNA methylation must be stably inherited and be instructive, for example by blocking the binding of activating transcription factors [11]. How much it is utilized in this way, however, remains open as the instructive potential and stability of DNA methylation appears highly dependent on the sequence context, in particular the local concentration of CpGs (reviewed in [11]). Furthermore, the impact of DNA methylation on the ability of individual transcription factors to bind DNA is largely undefined, which limits our ability to predict whether and where DNA methylation could be instructive and repressive. Loss of methylation can occur in a passive way by the absence of maintenance during cell division, but it can also occur actively. Such active, enzyme-catalyzed demethylation is present in the germline and early embryo [12], but also during somatic differentiation [13]. However, many proposed enzymatic pathways for active demethylation remained controversial [14] until the recent discovery of the 10–11 translocation (TET) family of proteins [15]. The TET proteins employ an oxidative mechanism that creates 5-hydroxymethylcytosine. This form of active demethylation furthermore creates an opportunity to identify sites of action as 5-hydroxymethylcytosine can be detected even at the level of individual bases [16,17]. TET proteins are expressed in nondividing somatic cells [18,19], suggesting that oxidation-dependent DNA demethylation happens in every cell type. The actual level and sites of active demethylation are currently under intense investigation. Their correct identification and proper quantification of the kinetics of DNA methylation turnover are critical not only for interpreting maps of DNA methylation but also for concepts of epigenetic memory that invoke DNA methylation. The latter require stability of the epigenetic mark and thus defining its local turnover is essential. Experimentally, this requires identifying sites of binding and measuring the local activity for both demethylases and de novo methyltransferases.

CpG islands The product of spontaneous deamination of the methylated cytosine is thymine, which is not recognized as a wrong base in DNA. This is different to uracil, the product of deamination of unmethylated cytosine, which is efficiently removed by repair enzymes. As a consequence, DNA methylation in the germline leads to a loss of the CG dinucleotide over evolutionary time. The result is an uneven distribution of CGs in mammalian genomes, which are largely devoid of CGs, which are concentrated within so-called CpG islands, regions of high CG density

that make up two out of three promoters in our genome. While CpG islands are efficiently repressed by DNA methylation (see below), it is less clear how these sequences are protected from being DNA methylated, and it has been suggested that this process requires both a local high concentration of the CG dinucleotide and the binding of transcription factors (reviewed in more detail in [11]).

Reading DNA methylation As the early days of its discovery, DNA methylation has been linked to gene repression [20]. Indeed, CpG-rich promoters (CpG islands) are efficiently repressed by DNA methylation, yet the underlying mechanism remains under debate [21,22]. Methylated promoters are occupied by methyl-CpG-binding domain (MBD) proteins in vivo [23,24]. These proteins are thought to repress transcription via recruitment of enzymes that deacetylate histones leading to a repressive chromatin state [25–27]. According to this indirect model of repression, DNA methylation-mediated silencing should be independent of sequences other than CpGs. Furthermore, the level of repression should increase with the local density of CpGs, which would explain why CpG islands are efficiently repressed if methylated. In addition to readers of 5-methylcytosine, there are also several reports of proteins recognizing 5-hydroxymethylcytosine and its further derivatives opening the possibility of specific readouts of these marks even if they occur in low numbers [28,29]. Alternatively, DNA methylation could repress in a direct way through blocking transcription factor binding. While such repulsion has been suggested for some factors, most prominently the insulator protein CTCF [30], it remains unresolved which transcription factors are inhibited by DNA methylation directly or indirectly. This is further complicated by the fact that some factors appear to differ in their methylation sensitivity dependent on the sequence context [31]. Alternatively, DNA binding factors could be attracted by their methylated motifs as suggested for KLF4, which could lead to locus-specific recruitment of repressors [29]. How transcription factors respond to local levels of methylation in vivo remains thus a crucial question in order to correctly interpret maps of DNA methylation. In the light of rapid recent advance in defining TF motifs [32], such information will hopefully be available in the near future.

Patterns of DNA methylation The majority of CpGs in vertebrate genomes are methylated, while unmethylated CpGs are concentrated in CpG islands (CGIs) [33,34]. The global loss of CpG dinucleotides in mammals is a consequence of DNA methylation itself as deamination of a methylated cytosine creates a thymine, which is not recognized by the DNA repair machinery, resulting in C to T

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transition [35]. The apparent high CpG density of CGIs is thus due to their hypomethylated state in the germline and the resulting absence of such mutations. CGIs make up the majority of gene proximal regulatory sequences as they overlap with about 60% of human and mouse promoters [36,37]. The importance of CGI promoter regulation is underlined by the fact that ~90% of mRNAs originate under their control [38]. While CpG-poor promoters are generally associated with tissue-specific expression patterns, many CGI promoters are active across various cell types and control promiscuously expressed ‘housekeeping’ genes [39]. Importantly, however, approximately 30% of CGI genes are tissue-specific and include developmental regulators such as the hox genes [36]. Most CpG-rich promoters are nevertheless devoid of DNA methylation also in differentiated cells [34]. The general absence of methylation at CGIs is thought to reflect their ability to block access for the de novo methylation machinery and/or the recruitment of mechanisms of active demethylation. Resistance to methylation could involve repulsion of DNMT3A and DNMT3B recruitment through histone modifications, as histones residing in nonmethylated CGIs are methylated at lysine 4 of histone H3 [40]. Furthermore, unmethylated CpGs can be bound by CXXC domain containing proteins (such as CFP1/Set1/MLL1/2), providing a potential positive feedback loop that reinforces the unmethylated state of CGIs [41]. Moreover, the unmethylated state could be reinforced by active demethylation as TET1 and TET3 both contain CXXC domains, which are important for correct targeting [42,43]. Interestingly, CGI methylation can become a frequent event in cancer illustrating loss of the underlying control in disease [44,45].

CpG-poor regions. One intriguing observation is that regulatory regions, which reside distal to promoters, show reduced levels of DNA methylation when they are active and occupied by transcription factors [54] (Fig. 1). This provides a general link between DNA methylation and enhancer activity and also explains part of the variability of DNA methylation that is observed between different cell types. At the same time, it remains to be determined how much reduced methylation of enhancers is part of their activation and thus instructive. The limited evidence thus far suggests that most of these dynamic changes occur subsequent of transcription factor binding [54– 56]. As already outlined above, further understanding how transcription factors respond to DNA methylation remains essential to answer this question. The observation that transcription factor binding can be frequently upstream of methylation directly predicts that genetic variations between individuals cause measurable variations in DNA methylation. For example, sequence variation within a binding site for a transcription factor would lead to reduced binding and in turn increased methylation. In such a case, methylation differences would reflect underlying genetic variation, and the epigenetic state would simply follow the DNA sequence [54,57]. The fact that enhancers show highly dynamic methylation provides a rich and unexpected source of information. Using

Genomewide maps of DNA methylation A variety of methods exist to detect DNA methylation. These range from the use of restriction enzymes that are sensitive to DNA methylation to affinity-based approaches that use antibodies or MBD domain proteins to enrich for methylated DNA [46–49]. The gold standard of analysis, however, remains bisulphite treatment, which converts unmethylated cytosines to uracil, but does not convert methylated cytosines. This enables detection by DNA sequencing at the resolution of single molecules and basepairs [50,51]. Recent advances in highthroughput DNA sequencing enable researchers now to use this approach genomewide [52]. Even though the sequencing efforts for such experiment are substantial and exceed those of genome sequencing, the recent drop in sequencing costs now enable to perform such experiments even at the level of patient cohorts [53]. Already the increased sensitivity and comprehensiveness of genomewide DNA methylation has revealed unexpected new insights into DNA methylation, particularly at


Figure 1 Graphic representation of observed DNA methylation patterns in mammalian genomes. CpG islands show no methylation and contain many CpGs, the remainder of the genome tends to be fully methylated except active regulatory regions that display reduced methylation as a function of their occupation by transcription factors ([54], figure adapted from [71]). The extent of the grey shadow represents the methylation variability of a given segment.

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only DNA methylation enables to identify enhancers and thus to reconstruct parts of cell specific gene regulation. For example, analysis of sites of demethylation at enhancers can help to identify the activity of cell-type-specific transcription factor motifs [58]. This has impact beyond the field of DNA methylation as it opens new avenues in diagnostics. As DNA methylation is readily accessible from patient DNA, it can be used as an important information proxy for gene regulation even when other molecular measures of regulatory activity such as RNA, chromatin, or the presence or local binding of transcription factors are not available.

Potential of DNA methylation as a biomarker Biomarkers are critically important not only in diagnostics but also to monitor treatment and drug efficacy. Better and more comprehensive detection is also a critical cornerstone towards ‘personalized medicine’. DNA methylation lends itself to diagnostics for technical reasons. The most established measurement method for DNA methylation is bisulphite conversion of unmethylated cytosines followed by DNA sequencing. This robust method can be performed with only little amount of DNA and has furthermore little quality requirements. As a result, bisulphite sequencing can even be performed on DNA isolated from fixed tissue [59]. It thus can be assumed that any primary sample that allows DNA sequencing is suitable for DNA methylation analysis. Next-generation DNA sequencing will be a key tool in future diagnostics and as a consequence all set-ups in terms of isolation and detection will be readily in place in laboratories that perform routine clinical diagnostics. Furthermore, bisulphite sequencing can be covered by a patient consent for DNA sequencing. As such technical issues for detection appear to be solved, the main question becomes how informative DNA methylation can be in a clinical setting. One argument against its utility is the fact that in many diseases, affected tissues are not available for testing and that DNA methylation, if measured from available cells such as PBMC, might be noninformative. Notably, however, many cases have been reported, where DNA methylation from blood samples can nevertheless be highly informative [60,61], utilized in treatment decisions [62,63] or definition of clinical cohorts [64] just to list a few examples. Genome methylation is already included as a measured variable in large cohort studies (e.g. [65–67].) and within large epigenome consortia [68] and thus we should soon have a more solid set of data to link phenotypes to DNA methylation. Selected genomewide association studies [69] already performed limited methylation analysis linked to transcription, so-called epigenomewide association (EWAS) [61,70]. These first cohort studies already revealed that DNA methylation mainly

follows DNA sequence, thus in many cases, there might be no additional information on top of DNA sequence variation. Beyond this dependence on DNA sequence, however, it can be expected that the measured epigenotype is more variable than the genotype as a reflection of disease penetrance. Thus, even though it is linked to a sequence variant, the methylation at some site might reflect actual phenotype manifestation and not only risk as the genotype. At this point, it is difficult to predict how informative and useful DNA methylation will become in the clinic. Given the revolution in DNA sequencing and the above outlined dynamics of DNA methylation, there is clearly sufficient potential to justify further exploration. Ongoing efforts in DNA methylation measurements in cohorts should be able to determine how much of the current excitement is justified. Address Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH 4058 Basel, Switzerland (D. Sch€ ubeler); University of Basel, Faculty of Science, Petersplatz 1, 4003 Basel, Switzerland (D. Sch€ ubeler). Correspondence to: Dirk Sch€ ubeler, Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH 4058 Basel, Switzerland. Tel.: +(41)616978269, Fax: (+41) 616973976; email: [email protected] Received 22 April 2014; accepted 15 January 2015 References 1 Bell O, Tiwari VK, Thoma NH, Schubeler D. Determinants and dynamics of genome accessibility. Nat Rev Genet 2011;12:554–64. 2 Ptashne M. On the use of the word ‘epigenetic’. Curr Biol 2007;17: R233–6. 3 Bird A. Perceptions of epigenetics. Nature 2007;447:396–8. 4 Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 2010;11:204–20. 5 Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 1998;19:219–20. 6 Bestor T, Laudano A, Mattaliano R, Ingram V. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J Mol Biol 1988;203:971–83. 7 Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247–57. 8 Bhutani N, Burns DM, Blau HM. DNA demethylation dynamics. Cell 2011;146:866–72. 9 Holliday R, Pugh JE. DNA modification mechanisms and gene activity during development. Science 1975;187:226–32. 10 Riggs AD. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet 1975;14:9–25.

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ESCI award lecture: regulation, function and biomarker potential of DNA methylation.

Methylation of DNA and modifications of histones have emerged as intricately involved in gene regulation as they cross-talk and respond in multiple wa...
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