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Enhancer DNA methylation: implications for regulation

Allegra Angeloni1, 2 and Ozren Bogdanovic1, 3

1Genomics and Division, Garvan Institute of Medical Research, Sydney, New South Wales, 2010, Australia 2St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, 2010, Australia. 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, 2052, Australia. Correspondence to: [email protected]

Abstract DNA methylation involves the addition of a methyl group to the fifth carbon of the pyrimidine cytosine ring (5-methylcytosine, 5mC). 5mC is widespread in vertebrate where it is predominantly found within CpG dinucleotides. In mammals, 5mC participates in long term silencing processes such as X inactivation, genomic imprinting, somatic silencing of germline , and silencing of repetitive DNA elements. The evidence for 5mC as a dynamic gene-regulatory mechanism is mostly limited to specific examples, and is far from being completely understood. Recent work from diverse model systems suggests that 5mC might not always act as a dominant repressive mechanism and that hypermethylated promoters and enhancers can be permissive to in vivo and in vitro. In this review we discuss the links between 5mC and enhancer activity, and evaluate the role of this biochemical mechanism in various biological contexts.

Introduction Chemical modifications to the DNA have the potential to transient and permanent changes in gene-regulatory activity that are heritable through cell division and through generations [1, 2]. In , the major form of DNA methylation involves the addition of a methyl group to the fifth position of the cytosine ring (5-methylcytosine, 5mC) by DNA methyltransferase enzymes (DNMTs) [3]. 5mC is established de novo by DNMT3A and DNMT3B and is maintained through DNMT1, which recognises hemi-methylated CpGs through the UHRF1 [4]. Maintenance of 5mC is essential to guarantee faithful re- establishment of 5mC on the newly synthesised strand following DNA replication. 5mC can be removed from the DNA through two major mechanisms: passive and active DNA demethylation. During passive DNA demethylation, 5mC is diluted following several rounds of DNA replication due to the absence of DNA methylation maintenance following replication (absence of DNMT1) [5, 6]. Conversely, in active DNA demethylation, 5mC can be removed enzymatically in a stepwise manner. Active DNA demethylation involves the iterative oxidation of 5mC by Ten-Eleven Translocation (TET) dioxygenase enzymes to the intermediates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) followed by their subsequent removal via the BER pathway [7]. In the animal kingdom, the predominant form of 5mC is usually found within CpG dinucleotides, however 5mC in CpH dinucleotides (where H is A, C or T) that is common in plant genomes, has also been identified in invertebrate genomes [8], and mammalian embryonic stem cells (ESCs) and brain tissue [9, 10]. The levels of 5mC in the CpG context can vary greatly between and within animal phyla [11, 12]. For example, in insects, genomic CpG methylation levels can range from 0% (Diptera) to 14% (Blattella asahinai - asian cockroach) [13]. Invertebrates on average display lower global 5mC levels than vertebrates, with the majority of 5mC localised to gene bodies of active genes [14]. Such intragenic 5mC has also been observed in highly transcribed genes in mouse ESCs where it is thought to prevent spurious transcription initiation as a result of cryptic entry by RNA polymerase II [15]. Unlike invertebrates, vertebrate genomes are characterized by high genomic 5mC that often reaches 70-80% [16]. Due to this high CpG 5mC content, CpG sites have been evolutionarily depleted from vertebrate genomes, which is a phenomenon that can be explained by increased deamination (C to T base change) of 5mC [17]. The exception to this global hypermethylated state are CpG islands (CGIs), short stretches of high CpG density that are mostly unmethylated and that often coincide with gene promoters [18]. Initial experiments that aimed to demonstrate the gene-repressive potential of 5mC involved the transfection of in vitro methylated templates to cultured cells and the subsequent measurement of their activity when compared to unmethylated templates [19, 20]. This was followed by the description of precise biochemical mechanisms of 5mC-mediated gene repression such as the recruitment of methyl-CpG-binding (MBD) and deacetylase (HDAC) containing complexes [21, 22]. These important observations resulted in speculations of 5mC potentially acting as a dynamic gene regulatory mechanism, with broad reaching implications for embryogenesis and cell differentiation. The advent of massively parallel sequencing technologies a couple of decades later allowed for unbiased insights into the dynamics of 5mC in diverse model systems. Collectively, this work unravelled that CpG island (CGI) promoters (i.e the majority of promoters in vertebrates) are by and large unmethylated independently of gene activity [23-25]. Those observations notwithstanding, it is important to note that when methylated, CGIs are strongly associated with transcriptional repression. For example, in both and mammals, a set of germline CGI promoters become methylated at early embryonic stages, resulting in permanent somatic silencing of germline genes [26, 27]. Distal regulatory elements such as enhancers display much greater differences in 5mC patterning between different tissues, cell types and developmental stages [28-30]. In general, enhancer activity as defined by (TF) binding is anti- correlated with 5mC content [31, 32]. However, the relationship between 5mC removal and enhancer activation is still not well understood. Here we discuss the functional links between 5mC and the activity of gene-regulatory elements, with a particular focus on transcriptional enhancers.

5mC and the regulation CpG island promoters CpG island (CGI) promoters are the most common type in vertebrate genomes. They are characterised by high CpG richness and elevated G+C content [18]. Notably, the majority of CGI promoters are unmethylated independently of gene activity, even though some CGI promoters become methylated and silenced during and X chromosome inactivation [26, 33, 34]. It is also worth noting that forceful methylation using targeted technologies does not result in permanent CGI 5mC and silencing [35]. The constitutively unmethylated state of CGIs is established and maintained by active histone marks, transcription factor binding, recruitment of CxxC domain proteins, and TET [36-43]. When methylated, CGIs can act as recruitment sites for MBDs such as MBD1, MBD2, MBD4, and MeCP2 [44]. These proteins appear to be constitutively bound to a selection of methylated CGIs in both pluripotent and differentiated states [44], in line with the absence of major 5mC changes during this developmental transition [33]. On the other hand, Polycomb group proteins display more extensive dynamics at CGIs during development and differentiation [33]. Overall, CGIs act as platforms for transcriptional regulation that are largely independent of 5mC dynamics. In the modest number of cases where 5mC is targeted to CGIs it appears that this process acts as an additional silencing safe-lock rather than the primary determinant of gene activity [26, 45].

5mC levels at enhancers Enhancers are traditionally defined as distal cis-acting regulatory elements that enhance transcription through recruitment of transcription factors (TFs) and co- proteins [46]. Enhancers activate and regulate expression of their target genes over distances that range from several hundreds of base pairs to a few megabases. In the absence of enhancers, transcription of associated genes is often weakened. On average, enhancers display low CpG density, even though CpG-rich enhancers have also been described [24, 28, 47, 48]. Based on their activity, enhancers can broadly be classified as: active, primed, poised, or silent. Active enhancers are characterised by depletion and the presence of active histone modifications such as and [46, 49]. Poised enhancers are depleted of and decorated with the permissive H3K4me1 and the repressive Polycomb H3K27me3 mark, whereas primed enhancers bear H3K4me1 and also display nucleosome depletion. Enhancers exhibit variable 5mC levels (10-50%) that are dependent on the extent of TF binding [31, 32]. An ENCODE study explored these trends globally by performing linear regression analysis between 5mC and accessibility (as measured by DNAseI hypersensitivity) at > 34,000 genomic sites, and revealed a significant association [50]. Increased 5mC was negatively correlated with chromatin accessibility at 20% of the examined sites. The same study compared TF expression levels to average 5mC levels at their corresponding binding sites across the . Similarly, a significant negative correlation was found between TF expression and 5mC content at TF binding sites for 70% of the TFs examined, arguing that 5mC deposition might be a direct consequence of the absence of TF expression [50]. Altogether, data from the studies discussed above suggest that active enhancers on average display less 5mC than poised or silent enhancers, yet their 5mC levels are not as low as 5mC levels observed in constitutively unmethylated CGIs [32].

5mC, 5hmC and TF-binding While TF-binding at enhancers is necessary for transcriptional regulation of the associated genes, it is not yet established whether TF-binding induces the formation and maintenance of hypomethylation at enhancers. A recent high throughput study examined the effects of 5mC on TF binding and found that the majority of TFs assayed (33%) were not repelled by 5mC, whereas 34% and 23% of TFs displayed increased and decreased binding to methylated sequences, respectively [51] (Fig. 1a). Moreover, recent work has demonstrated that 5mC as well its oxidative intermediates: 5hmC, 5fC, and 5caC can recruit transcriptional activators in vitro [52]. The protein CTCF has been studied extensively in terms of its interactions with 5mC. Data from mouse embryonic stem cells (ESCs) suggest that CTCF can mediate active DNA demethylation at bound regions [31] and that the introduction of CTCF sites in stably-integrated reporter constructs results in decreased 5mC [32]. Similarly, reintroduction of TFs is able to reverse a hypermethylated regulatory region to a hypomethylated state once bound by a TF. This has been demonstrated in mouse ESCs that lack the transcription factor REST, in which REST-/- cells display increased 5mC and reduced 5hmC at REST binding sites compared to wild-type cells. Reintroduction of REST leads to decrease in 5mC and increase in 5hmC at binding sites, indicative of an active, TET-dependent mechanism of demethylation [31]. This is further supported by studies of the glucocorticoid receptor binding to tissue-specific enhancers, in which binding of the receptor induces DNA demethylation resulting in enhancer activation [53]. Given the modest number of reports describing such examples, it is currently difficult to propose a unifying molecular mechanism of TF-mediated demethylation of regulatory regions. It is likely however that TET methylcytosine dioxygenases play a role in at least a subset of such interactions (Fig. 1b,c). Enrichment of 5hmC, the major 5mC oxidised derivative, has been identified on predicted enhancers in mammalian ESCs [54, 55]. In human ESCs, 5hmC-enriched enhancers coincide with binding sites of pluripotency regulators such as NANOG and OCT4 [54] and TET2 knockouts in mouse ESCs result in extensive loss of 5hmC at enhancers, accompanied by 5mC enrichment (hypermethylation) and reduction of enhancer activity [55]. This is indicative of robust 5mC oxidation and TF turnover on key regulatory elements associated with mammalian pluripotency. It is also worth noting that DNMT1 has low binding affinity for 5hmC, therefore 5hmC may further promote passive DNA demethylation by excluding DNMT1 and thus preventing methylation of cytosines on the nascent DNA strand [56]. Mechanistically, 5hmC has been shown to disrupt nucleosome stability in vitro, and to decrease binding affinity of a major transcriptional , MeCP2 [57, 58]. Taken together, this suggests that 5hmC might contribute to enhancer activity by promoting both active and passive DNA demethylation and by influencing chromatin structure.

Enhancer methylation and embryonic development Once established in adult tissues, 5mC is a relatively static mark. However, during early mammalian embryogenesis it undergoes two rounds of global reprogramming. The first wave of genome-wide demethylation occurs shortly after fertilisation whereas the second wave takes place in primordial germ cells (PGCs) [1, 2]. While such global reprograming appears to be a mammalian-specific phenomenon, active DNA demethylation at distal regulatory elements during vertebrate body plan formation is an evolutionarily conserved trait. A wave of active DNA demethylation at thousands of enhancers associated with conserved regulatory pathways such as Wnt, Notch and transforming growth factor (TGF)-β results in coordinated transcriptional changes and morphological similarities during the vertebrate phylotypic period, the most highly conserved stage of vertebrate embryogenesis [28]. The necessity of this epigenetic remodelling event is highlighted through triple TET zebrafish morphants, which display embryonic lethality and severe developmental including short and blended axes, reduced pigmentation and impaired head structures, and which coincide with increased 5mC at phylotypic stage enhancers. Notably, these ~ 2500 phylotypic stage enhancers are highly CpG rich, displaying a CpG density comparable to that of CGIs, which might make them more prone to regulation via 5mC. Importantly, triple TET morphants also display reduced chromatin accessibility at phylotypic enhancers, as measured by ATAC-seq, indicative of 5mC being an upstream regulatory mark, the removal of which might be required for proper enhancer function. Similar impact on chromatin accessibility at regulatory regions has been demonstrated in mammalian systems where TET2/TET3 function was ablated [59, 60]. Studies of mouse and human organogenesis during early embryogenesis have also revealed a conserved and elaborate methylation reprogramming of enhancers strongly associated with the development of almost all human organs, displaying the canonical inverse correlation between 5mC levels and enhancer activity [61]. Moreover, recent work suggests that developmental removal of enhancer methylation might have preceded the origin of vertebrates. The invertebrate chordate Amphioxus (Branchiostoma lanceolatum) has been shown to display robust demethylation of regulatory regions (as defined by ATAC-seq) during tissue differentiation [62]. Thus, evolutionary conservation of developmental enhancer demethylation and the expression of associated developmental genes, appears to be a feature required for proper embryonic development of vertebrates and beyond.

Compatibility of 5mC and active chromatin signatures Despite the insights discussed above, the relationships between 5mC levels and enhancer activity are far from being fully understood. For example, in mouse genomes, distal regulatory elements display tissue-specific differential methylation. A subset of these differentially methylated regions is hypomethylated yet lack the active histone marks H3K4me1 and H3K27ac, and are consequently inactive in adult tissues. However, these enhancers ("vestigial" enhancers) are active during embryonic development, displaying hypomethylation as well as H3K4me1 and H3K27ac, with their putative targets being developmental genes, suggesting that a form of “epigenetic memory” is retained from development to adulthood in the form of DNA hypomethylation [29, 63]. The methylation status of an enhancer can therefore be utilised as an indicator of the past activity of an enhancer. More notably however, these findings demonstrate that hypomethylation at these regions is not necessary or sufficient for enhancer activity. Moreover, a recent study exploring the correlation between chromatin structure and 5mC has challenged the typical inverse relationship between 5mC content and enhancer activity, demonstrating that 5mC can co-exist with active histone marks at distal regulatory sequences [64]. This report suggests that chromatin can exist in a bivalent state, characterised by the coexistence of both active histone marks such as H3K27ac and 5mC in human cells. Specifically, high levels of 5mC were detected on the same nucleosome with H3K27ac marks at regular and super-enhancers in the , co-existing in an unprecedented relationship that is not observed at promoters. Moreover, some of these regions were highly CpG dense, displaying CpG content comparable to that of CGIs. As these bivalent regions appear to be located outside TF- binding sites in most of the cases, it is possible that 5mC in this context does not repress enhancer activity, but rather stabilises DNA and defines demethylated TF-binding sites [64]. A similar observation regarding the compatibility of 5mC and active histone marks was also recently made in zebrafish embryos [65]. These observations notwithstanding, it is important to note that the majority of vertebrate enhancers are CpG poor and that it is not clear what impact 5mC has on their activity. Despite the compelling evidence suggesting that 5mC state plays an important role in determining enhancer activity, whether there is a strict causal relationship between methylation and enhancer activity remains unresolved.

Conclusion While 5mC is one of the most well studied epigenetic modifications, the functional consequence of this mark in certain genomic contexts remains enigmatic. The significance of 5mC at distal regulatory elements is augmented by the fact that it is an evolutionarily conserved feature that must be carefully orchestrated for normal embryonic development and cell differentiation. Evidently, a number of studies support that enhancer methylation influences transcription of the associated gene, however there is currently a lack of a definitive causal link that explains whether 5mC is directly responsible for enhancer activity. Furthermore, the chromatin environment that is necessary for 5mC to influence is not entirely understood. Studies performed in diverse biological systems typically suggest that active enhancers display lower 5mC levels than poised or silent enhancers, as well as TF binding, 5hmC enrichment and the presence of active histone marks H3K4me1 and H3K27ac [32]. Conversely, hypermethylation at enhancers is generally associated with reduced chromatin accessibility and decreased TF binding. This consensus has been challenged by recent studies on the epigenetic environment of enhancers. Enhancers can exist in a bivalent state, displaying the co-existence of characteristic active and inactive biochemical features [66]. It is also important to note that enhancer CpG density might play a major role in determining the regulatory inputs and outputs of enhancers. Thousands of CGI enhancers identified in human tissues display considerable regulatory differences when compared to their CpG poor counterparts. For example, CGI enhancers are: i) more highly conserved, ii) more highly enriched in R loops and 3D genomic contacts, iii) on average provide stronger signal in functional assays, and iv) are prone to hypermethylation in cancer [47]. Future work involving technologies such as precise epigenome editing [67] and single cell genomic and transcriptomic assays should aim to conclusively define how and to what extent 5mC presence at distinct enhancer classes is necessary for interaction with the linked promoter and consequently for the control of gene expression.

Summary

• In general, at least in vertebrates, enhancer activity is correlated with DNA hypomethylation. Some studies, however, suggest that 5mC and active chromatin marks such as H3K27ac are compatible at enhancers. • DNA hypomethylation is not sufficient to drive enhancer activity as evidenced by embryonic enhancers that remain hypomethylated and yet are inactive in adult mammalian tissues. • A number of studies have shown that TF binding to enhancers is sufficient to promote 5mC turnover that can be mediated by TET-dependent mechanisms of active demethylation. • Developmental 5mC remodeling at enhancers appears to be a conserved feature of vertebrate and perhaps even chordate and to some extent invertebrate development.

Conflicts of interest The authors declare no conflict of interest associated with this manuscript.

Acknowledgments This work was supported by the ARC Discovery Project (DP190103852) to O.B. The figures were made with the help of Biorender software.

Author contributions A.A and O.B wrote the paper. O.B drafted the figures.

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Figure 1. 5mC turnover at gene-regulatory regions. a) 5mC sensitivity of > 500 TFs assayed through methyl-SELEX and bisulfite-SELEX approaches [51]. The majority of TFs were not affected by 5mC whereas more than 30% of assayed TF displayed increased affinity toward methylated motifs. This was followed by TFs that displayed reduced affinity towards 5mC (> 20%). A small percentage of TFs displayed minor or multiple effects. b-c) Schematic examples of cell lines where a transcription factor (TF) is silenced (b) or expressed (c). Work from multiple sources [31, 32, 50] suggests that TF occupancy anti-correlates with 5mC at regulatory regions and that forceful expression of TFs is sufficient to induce a hypomethylated state at TF binding sites, likely through the recruitment of TET demethylases.

Figure 2. Non-canonical chromatin configuration at enhancers. a) Vestigial enhancers [29] are embryonic enhancers that remain hypomethylated in adult tissues, however, without displaying any active chromatin marks. It is unclear to what extent TF binding participates in the formation of this type of chromatin and what the function of such "epigenetic memory is". b) Bivalent enhancers display active chromatin marks such as H3K27ac ad 5mC on the same nucleosome. Such enhancers also encompass super-enhancers (enhancer clusters). It is postulated that the combination of H3K27ac and 5mC participates in enhancer integrity and the definition of TF binding sites.