REVIEW Role of TET enzymes in DNA methylation, development, and cancer Kasper Dindler Rasmussen1,2 and Kristian Helin1,2,3,4 1Biotech Research and Innovation Centre (BRIC), 2Centre for Epigenetics, 3The Danish Stem Cell Center (Danstem), 4Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark The pattern of DNA methylation at cytosine bases in the maintain cell type-specific transcriptional programs. genome is tightly linked to gene expression, and DNA Along with post-translational modifications of histone methylation abnormalities are often observed in diseases. proteins, the direct methylation of cytosines in CG dinu- The ten eleven translocation (TET) enzymes oxidize 5- cleotides in DNA (called CpG sites) is one of several layers methylcytosines (5mCs) and promote locus-specific re- of regulatory information that determines chromatin versal of DNA methylation. TET genes, and especially states. Until recently, DNA methylation was believed to TET2, are frequently mutated in various cancers, but be an irreversible epigenetic event associated with gene how the TET proteins contribute to prevent the onset repression, which could only be alleviated through DNA and maintenance of these malignancies is largely un- replication. Thus, it was remarkable when ten eleven known. Here, we highlight recent advances in under- translocation protein 1 (TET1) was discovered and shown standing the physiological function of the TET proteins to be able to modify methylcytosine and potentially erase and their role in regulating DNA methylation and tran- DNA methylation (Tahiliani et al. 2009). TET1 belongs to — scription. In addition, we discuss some of the key out- a family of three proteins namely, TET1, TET2, and — standing questions in the field. TET3 that catalyze the successive oxidation of 5-meth- ylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) (He et al. 2011; Ito et al. 2011). These 5mC oxidation prod- Pertinent questions ucts were promptly implicated as intermediates in the conversion of 5mC to unmodified cytosines, potentially • How are TET proteins recruited to genomic elements? providing the first steps in a pathway for active DNA • Does deposition of 5-hydroxymethylcytosine (5hmC) demethylation and implying that DNA methylation pat- inhibit the maintenance of DNA methylation in vivo? terns are not as static as previously assumed. Disruption of epigenetic landscapes, including DNA • Do 5hmC/5-formylcytosine (5fC)/5-carboxylcytosine (5caC) methylation patterns, is a hallmark of cancer. Somatic have roles as epigenetic marks? mutations in genes (e.g., in DNMT3A) that encode for • What is the link between TET2 inactivation, the machinery that establishes DNA methylation have transcriptional changes, and premalignant and malignant been causally linked to malignant transformation. Inter- hematopoiesis? estingly, the activity of TET enzymes, which is involved • Are fully transformed tumor cells dependent on the initial in removing this epigenetic mark, has also emerged as TET mutation? an important tumor suppressor mechanism in cancer. TET2 is one of the most frequently mutated genes in he- • Do the biological functions of the TET proteins depend on matopoietic malignancies, and its disruption is an early their catalytic function? event in the onset of disease. It is now known that all three TET genes are mutated and show reduced expression, and the proteins have impaired activity in a wide range of dif- The vertebrate genome is bundled into a highly organized ferent cancer types. Thus, precise regulation of DNA chromatin structure fundamental for precise gene regula- methylation patterns, which is partly mediated by TET enzymes, is important for normal development tion and maintenance of genome integrity. It is widely be- and provides a fundamental protection against cellular lieved that dynamic chromatin states guide cells through transformation. development and impart an epigenetic “memory” to © 2016 Rasmussen and Helin This article is distributed exclusively by [Keywords: DNA methylation; DNA demethylation; TET; Cold Spring Harbor Laboratory Press for the first six months after the hydroxymethyl cytosine; hematopoiesis; leukemia] full-issue publication date (see http://genesdev.cshlp.org/site/misc/ Corresponding author: [email protected] terms.xhtml). After six months, it is available under a Creative Commons Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.276568. License (Attribution-NonCommercial 4.0 International), as described at 115. http://creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 30:733–750 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/16; www.genesdev.org 733 Rasmussen and Helin TET proteins and their role in regulation of DNA DNA methylation patterns across cell divisions and is be- methylation patterns lieved to result in replication-dependent dilution of 5mC. In addition to down-regulation or cytoplasmic retention of TET proteins are large (∼180- to 230-kDa) multidomain the DNA methylation machinery, the deposition of enzymes (Fig. 1). All TET proteins contain a conserved 5hmC can have a role in inducing passive DNA demethy- double-stranded β-helix (DSBH) domain, a cysteine-rich lation. In vitro studies have shown that DNMT1 activity domain, and binding sites for the cofactors Fe(II) and 2- is dramatically reduced (up to 60-fold) on a DNA substrate oxoglutarate (2-OG) that together form the core catalytic containing 5hmC (Valinluck and Sowers 2007; Hashi- region in the C terminus. Structural studies suggest that moto et al. 2012; Ji et al. 2014). TET-mediated 5hmC this core catalytic region preferentially binds cytosines deposition may therefore trigger passive replication-de- in a CpG context but does not interact with surrounding pendent DNA demethylation on the opposite DNA strand DNA bases and shows little or no specificity for flanking and be important to counter sporadic accumulation of ab- DNA sequences (Hu et al. 2013, 2015; Hashimoto et al. errant DNA methylation. However, more recent data sug- 2014b). In addition to their catalytic domain, TET1 and gest that the role of 5hmC could be more complex. It has TET3 have an N-terminal CXXC zinc finger domain been shown that the DNMT1 interaction partners UHRF1 that can bind DNA (Fig. 1; Zhang et al. 2010; Xu et al. and UHRF2 can bind 5hmC DNA (Frauer et al. 2011; 2011c, 2012). Hashimoto et al. 2012; Iurlaro et al. 2013; Spruijt et al. The deposition and removal of DNA methylation occur 2013; Zhou et al. 2014). It is therefore possible that one in a cyclical manner and require the enzymatic activity of or both of these promote maintenance of DNA methyla- multiple proteins. The majority of CpG sites are symmet- tion by recruiting DNMT1 to hemihydroxymethylated rically methylated such that both strands carry the cyto- sites in vivo. Moreover, the two “de novo” DNA methyl- sine modification. During S phase, the SRA domain of transferases DNMT3A2 and DNMT3B2 have also been UHRF1 recognizes and binds newly replicated hemime- implicated in the maintenance of DNA methylation thylated DNA (Jones and Liang 2009). This interaction (Chen et al. 2003; Jones and Liang 2009), and these en- promotes recruitment of the “maintenance” DNA meth- zymes readily methylate a DNA substrate containing yltransferase DNMT1 that re-establishes symmetry by 5hmC (Hashimoto et al. 2012; Ji et al. 2014). Widespread copying the DNA methylation pattern of the template deposition of 5hmC has also been proposed to induce pas- strand onto the newly synthesized strand. This mecha- sive DNA demethylation in preimplantation embryos and nism is responsible for faithful inheritance of the bulk of primordial germ cells (for review, see Hill et al. 2014). DNA methylation patterns across cell division (Jones However, it is now becoming apparent that the global and Liang 2009). Once methylated, the modified cytosine demethylation observed in these cell types can proceed in- can be the substrate of a stepwise TET-mediated oxidation dependently of 5hmC production. For instance, DNMT1 process that forms 5hmC, 5fC, and 5caC in a Fe(II)- and 2- is excluded from the nucleus in early embryos (Seisen- OG-dependent manner. In contrast to 5mC, the majority berger et al. 2013), and the rate of demethylation of both of these oxidation products is asymmetrically deposited paternal and maternal genomes cannot be explained by a on a given CpG site, suggesting that mitotic inheritance, passive demethylation pathway (both TET-dependent if any, is achieved by a different mechanism (Jones and and TET-independent) alone (Wang et al. 2014). Thus, Liang 2009; Yu et al. 2012; Wu et al. 2014). the overall contribution of 5hmC to global demethylation The conversion of 5mC and its oxidized derivatives processes is still not fully understood (Hill et al. 2014), and back to the unmodified state has been proposed to occur further investigations are necessary to discern the effect of by either “passive” or “active” demethylation. “Passive” 5hmC on the maintenance of DNA methylation in vivo in DNA demethylation refers to the failure to maintain cells with intact DNA methylation machinery (Fig. 2A). Core catalytic domain Figure 1. Domain structure of TET proteins. The C-terminal core catalytic domain shared by all TET TET1 (2136 aa) N C enzymes consists of the DSBH domain, a cysteine- rich (Cys) domain, and binding sites for the Fe(II) and 2-OG cofactors. The DSBH domain contains a large low-complexity region of unknown function. TET1 and TET3 have an N-terminal CXXC domain that TET2 (2002 aa) N C can bind directly to DNA and facilitate recruitment to genomic target sites. TET3 (1660 aa) N C Low complexity region Fe(II) 2-OG CXXC Cys DSBH binding binding site site 734 GENES & DEVELOPMENT TET proteins and DNA methylation “Active” DNA demethylation refers to an enzymatic zyme that binds and excises mismatched pyrimidines in process in which 5mC bases, also in their oxidized forms, G:U and G:T base pairs (Lindahl and Wood 1999). Subse- are replaced by unmodified cytosines in a replication-in- quent to the discovery of oxidized 5mC bases, it was dem- dependent manner.