Introduction

DNA methylation is one of the most common DNA modifications in mammalsand typically occurs at the CpG dinucleotide site where a methyl group is added to the fifth position of cytosine to generate 5-methylcytosine.1,2,3,4 This process is mediated by DNA methyltransferase (DNMTs). Among themDNMT3aDNMT3band DNMT3c establish de novo methylation by targeting unmethylated CpG siteswhile DNMT1 predominantly serves as a maintenance methyltransferase during cell divisions.5,6 Although DNA methylation is generally stableit can be removed by active demethylation associated with TET dioxygenases (DNA replication-independent) and passive demethylation (DNA replication-dependent). TET dioxygenasesspecificallyTET1TET2and TET3 oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)5-formylcytosine (5fC)and 5-carboxycytosine (5caC) in an Fe (II)/α-ketoglutarate-dependent manner.7,8,9 Notably5fC and 5caC can be excised by thymine-DNA glycosylase (TDG)and the modified site returns to the unmethylated status through base excision repair (BER).10,11,12,13,14 Thereforethese enzymes regulate active turnover of DNA methylation. BesidesUHRF1 recognizes 5mC:C dyads and recruits DNMT1 to hemi-methylated CpG sites to maintain DNA methylation.15,16 Disruption of this DNA methylation machinery dilutes 5mC during DNA replication. In addition5hmC reduces the affinity of UHRF1 towards 5hmC:C dyads and alters the specificity of DNMT1.17,18,19 Additionally5fC:C and 5caC:C dyads are capable of reducing the activity of DNMT1 in vitro.20 These observations suggest that all three oxidation products of TETs (5hmC5fCand 5caC) are poor DNMT1 substrates and are involved in passive DNA demethylation.

TET1 was the first identified member of TET familyacting as a fusion partner of MLL gene in acute myeloid leukemia patients bearing the t(10;11)(q22;q23) translocationand TET2 and TET3 were subsequently identified based on their significant sequence homology to TET1.21,22 The biological function of TET family was unclear until two landmark discoveries by Kriaucionis et al. 23 and Tahiliani et al. 7. They found TET1 could covert 5mC to 5hmCwhich was an Fe (II)/α-ketoglutarate-dependent enzyme by homology searching for JBP1known as enzymes to oxidize methyl-thymine.7,24,25 Further findings revealed that TET2 and TET3 also could catalyze similar reactions.8 In addition to converting 5mC to 5hmCTETs were capable of oxidating 5hmC to 5fC and further to 5caC.9

The mechanism underlying TET-mediated demethylation of DNA was not clear until 2011when two important papers identified that the oxidization products of 5mC5fCand 5caCcould be excised by TDG,10,11 suggesting that TET-mediated oxidization was implicated in active DNA demethylation26,27,28 This was supported by the following study that biochemical reconstitution of TET-TDG-BER system could lead to DNA demethylation29(Fig. 1a).

Fig. 1
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Function and structure of TET proteins. a The dynamic cycle of DNA methylation and demethylation. DNA methyltransferases (DNMTs) catalyzed the formation of 5-methylcytosine (5mC)which can be removed by TET-mediated oxidationcoupled with thymine-DNA glycosylase (TDG)-involved excision and base excision repair (BER). b Domain structure of TET proteins. All TET proteins possessed one core catalytic domain in C-terminal. A CXXC domainlocated in the N-terminal of TET1 and TET3but not in TET2conferred DNA binding ability directly

Because DNA demethylation and the 5hmC mark involve in various biological reactionsTETs play a very important role in both physiological and pathological processeswhich have been elucidated by many studies.13,30,31,32,33,34,35,36 For exampleTET2 loss resulted in hypermutagenicity in haematopoietic progenitor cellsunveiling a key role of TET2 in safeguarding cells against genomic mutagenicity.37 Dysfunctions of TET2 in cancer are associated with TET2 mutation and abnormal expression of TET2 regulators.32,38,39,40,41,42 Of notesince 2009many studies have demonstrated that TET2 mutations were frequently identified in multiple hematologic diseases.43,44,45,46,47,48,49 By genomic sequencingone study revealed that TET2 mutations were present in 14% (2 of 14) of patients with myelodysplastic syndrome (MDS)37% (11 of 30) with myelodysplastic/myeloproliferative neoplasms (MDS/MPN)and 43% (6 of 14) with secondary acute myeloid leukemia (sAML) evolved from MDS/MPN. Among the patients harboring TET2 mutationsMDS/MPN accounted for 58% (11 of 19)sAML evolved from MDS/MPN represented 32% (6 of 19)and MDS comprised 10% (2 of 19).44 HoweverTET2 mutations were infrequent in patients with solid tumors,50 despite somatic mutations in TET1(8 of 74)TET2(5 of 74)and TET3(4 of 74) were identified in colon cancer.51 Other molecular mechanisms underlying the dysregulation of TETs functions in both blood and solid cancers were diverse and complex such as metabolic alterations.52,53 These are discussed in the part of TETs function regulators.

In the following sectionswe discuss the structuresfunctionsand regulators of TETs and summarize the representative methods for 5hmC detection and epigenetic editing.

TET family structure

The primary structure of TETs contains a carboxy-terminal catalytic domainwhich is made up of a cysteine-rich domain (CRD)and two double-stranded β-helix (DSBH) regions separated by a large low-complexity insert.7,25 The DSBH domain possesses key residuesresponsible for binding to its cofactors (α-ketoglutarate and Fe (II))which are necessary to its catalytic function.54 Two zinc fingers combine the DSBH and CRD together to form the compact catalytic core.54 Although TET proteins are capable of oxidizing 5mC to 5hmC5fC and 5caCstructure analysis revealed that TET2 preferred 5mC substraterather than 5hmC and 5fC.55

TET1 and TET3 contain a CXXC domainlocated in the amino-terminal regionwhich is implicated in binding to CpG dinucleotides,56,57 whereas TET2 loses its CXXC domain likely due to a chromosomal inversion (Fig. 1b). Consequentlythis allows the ancestral TET2 CXXC domain to be a separate gene called IDAX (also named CXXC4). In the case of TET1 and TET3 with their respective CXXC domainsthey can bind with DNA directly. In vitro binding assays revealed that TET1 slightly preferred substrates of unmethylated over that of methylated.58 Further studies showed thatsimilar to other proteins harboring the CXXC zinc finger domainTET1 preferentially bound to CpG-enriched promoters of geneswhich was certified by chromatin immunoprecipitation of TET1 coupled with DNA sequencing in mouse embryonic stem cells (mESCs).59,60 SimilarlyTET3 CXXC-bound regions exhibited a significant enrichment of CpG and more than half of them were enriched in gene promoters.57 This study further revealed that the CXXC domain of TET3 was essential to its biological function through biochemical and structural analysis.57 In contrast5hmC regulated by TET2 is mainly located in gene bodies and exons rather than gene promoters.61 Of noteTET1/3 can be also recruited by their binding proteins for context-specific DNA regions.62,63 For examplethe pluripotency factor NANOG interacted with TET1 and ChIP-seq analysis identified TET1-NANOG co-binding sites associated with NANOG target genessuggesting that NANOG guided TET1 to specific sites of chromatin and some DNA-binding proteins were also important to TET1 functions.62

TETs functions and binding partners

The primary functions of TETs are able to oxidate 5mCand the products are subsequently involved in DNA demethylation.64,65,66 BesidesTET genes expression in different tissues are analyzed using the proteinatlas database67(https://www.proteinatlas.org/) in Fig. 2which may suggest unique and various functions of TETs in tissues. Evidences also support 5hmC as an epigenetic marknot only a demethylation intermediate.68 In additionthe non-catalytic activities of TETs are discovered.69 In this sectionwe discuss the classical and non-classical functions of TETs (Fig. 3). As TETs binding partners appear to be their main regulatorswe also summarize their information here (Tables 1–3).

Fig. 2
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Expression levels of TET1(a)TET2(b)and TET3(c) in different tissues. The data were obtained from the proteinatlas.67 Modified from images available for TET1 (https://www.proteinatlas.org/ENSG00000138336-TET1/tissue)for TET2 (https://www.proteinatlas.org/ENSG00000168769-TET2/tissue)and for TET3(https://www.proteinatlas.org/ENSG00000187605-TET3/tissue). nTPM: consensus normalized expression

Fig. 3
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TETs working models. a A particular transcription factor (TF) recruited TETs to specific DNA areas of promoters to increase the downstream gene expression in a dioxygenase activity-dependent manner.100,120 b TETs binding with other epigenetic regulatory enzymessuch as HDACtogether regulated the particular gene expression independent of TETs enzyme activity.183,187 cd TET1/2 oxidated mRNA (c)191,194 and TET2 oxidated tRNA (d)196 to exhibit regulatory functions in RNA levels

Table 1 Different types of TET1 binding proteins and their functions
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Table 2 Different types of TET2 binding proteins and their functions
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Table 3 Different types of TET3 binding proteins and their functions
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Classical functions of TETs

TET1

Studies have shown the important role of TET1 in physiological functionsincluding development.70 The deficiency of TET1 lowered female germ-cell numbers by controlling meiosis through mediating related-gene DNA demethylation,71 while TET1 loss was dispensable for mice embryonic and postnatal development.72 Howeveracute deletion of TET1 caused a significant decrease of 5hmC levels and impaired embryonic stem cell identity,73 possibly because a long-term chronic reduction in TET1 led to homeostatic compensation.73 TET1 is also essential for intestinal stem cell functions in vivo74 and implicated in dynamic changes of DNA methylation during the maturation of fetal intestinal epithelial organoids in vitro.75 Epigenetic programming by catalytic-dependent TET1 is implicated in liver regeneration76 and remyelination in mouse brain.77,78 TET1 is involved in regulating iron homeostasis by demethylating the promoter of RNF217 and this ubiquitin ligase is responsible for the degradation of iron exporter ferroportin.79 TET1-deficient mice exhibit impaired spatial learning and memory.80 Findings also support the roles of TET1 in promoting pluripotent stem cell induction.81,82,83,84 In additionTET1 is required in the reprogramming of fibroblasts to dopaminergic neurons.85

Abnormal expression of TET1 is associated with many diseases.86,87,88,89,90,91,92 Loss of TET1 led to B cell malignancy in aged micepartly due to aberrant DNA-hypermethylation,93 although TET1 acted as an oncogene in MLL-rearranged leukemia.94 Additionallyinsufficient TET1 was implicated in pulmonary arterial hypertension.95 Interestinglyoverexpression of TET1 promoted cholangiocarcinoma progress via proliferative and anti-apoptotic signaling pathways,96 while insufficient TET1 accelerated intestinal tumorigenesis.97 Besideshigh expression of TET1 appeared to be involved in polycystic ovary syndrome with hypomethylation signatures.98

Protein interactions enable rapid regulation and represent an important regulation in TETs functionswhich allow precise modifications in specific DNA loci timely.99 For exampleTET1 interacted with STAT1contributed to the demethylation of IRF1 promoter and transcriptional upregulation of PD-L1to drive tumor immune evasion.100 In additioninterestinglyFXR1an m6A readerguided TET1 to specific genomic loci near m6A RNA to result in DNA demethylationrevealing a novel regulation between RNA modification and DNA demethylation.101 Similar models have been supported by many findingswhich are summarized in Table 1.

TET2

Unlike TET1 and TET3TET2 mutations with high frequency are identified in hematologic malignancies.102,103,104,105 Thusthe relationship between TET2 mutations and overall survival has been investigated. Evidence showed that the patients with TET2 mutations had worse overall survival compared with the patients with wild-type TET2 in 93 patients with AML.38 Howeverother studies showed no survival association in 111 patients with de novo AML106 and in a cohort of 247 patients with secondary AML.107 Thereforethe significance of TET2 mutations in AML prognosis remains to be elucidated. The effects of TET2 mutations on its functionssuch as enzymatic activity and the ability of binding other proteinsand potential confounding variables should be considered. Further studies suggest that TET2 works as a tumor suppressor.108,109,110,111,112 Interestinglyfindings also reveal tumor-promoting roles of TET2.113,114 For exampleTET2 maintained the immunosuppressive-related gene expression in tumor-associated macrophages.115

As TET2 does not contain the CXXC domainthis raises a question of how TET2 is bound with the chromatin? One reasonable hypothesis is thatIDAXoriginating from the ancestral TET2 CXXC domainmediates its chromatin recruitment. Indeedbiochemical studies demonstrated that IDAX could bind to TET2 directlysuggesting that IDAX was able to recruit TET2 to DNA.116

In addition to IDAXsome other TET2-binding proteins have been identified by biochemical studies (Table 2). For instanceTET2 interacted with NANOG and played an important role in the establishment of pluripotency in a NANOG-dependent manner.117 EBF1a transcription factorhad also been identified as a TET2-binding protein by co-immunoprecipitation of TET2 and EBF1. Importantlysequence analyzing revealed that these two proteins were enriched in a proportional wayimplying that TET2without a CXXC DNA-binding motifexploited a DNA-binding proteinsuch as a transcription factorto regulate sequence-specific DNA demethylation.118 This idea was reinforced by the interaction of TET2 with WT1.119,120 Further studies revealed that mutations of TET2 and WT1 were mutual exclusively in AMLand WT1 guided TET2 to a specific DNA sequenceleading to the demethylation and activation of WT1-target genes.120 Many following studies supported this modelin which a DNA-binding factor recruited TET2 to a specific DNA sequence and regulated the expression of this gene in certain contexts.121,122,123,124,125,126,127 For instancewe found that the transcription factor HNF4α could recruit TET2 to FBP1 promotersresulting in the increase of FBP1 expressionto suppress the tumor growth.127 These models relied on the oxygenase activity of TET2.

TET3

As a member of the TET familythe main role of TET3 is implicated in demethylation in many biological processes such as zygote formation,128,129,130,131 embryogenesis,132 axon regeneration,133 and synaptic transmission.134 For exampleTET3-mediated DNA demethylation is necessary for liver tissue maturation via proper hepatocyte gene expression.135 In additionTET3 deficiency induced by mutations is associated with abnormal growth and intellectual disability,136 indicating the fundamental role of TET3 in development. In adult miceTET3 ablation is associated with anxiety-like behaviorsalthough the molecular mechanisms remain to be explored.137

Interestinglyhepatic TET3 was recruited to the promoters of the fetal version of HNF4α by FOXA2contributing to high expression of HNF4α transcription by promoter demethylationand this process impaired glucose homeostasis due to HNF4α-mediated gluconeogenesis activation. Thusthese findings linked TET3 to type-2 diabetes.138 In additioninsufficient demethylation of several insulin secretion genesowing to the maternal inheritance of oocyte TET3 insufficiencycontributed to glucose intolerance.139 These findings demonstrated the distinct roles of TET3 in certain contexts. Similar to TET2binding partners are involved in TET3 function regulation (Table 3). For examplePGC7 interacted with TET3 and suppressed TET3 enzymatic activity to protect DNA methylation at imprinting loci during early embryogenesis,140 although PGC7 bound to H3K9me2 to block the TET3-mediated conversion of 5mC to 5hmC.141

TET1/2/3

Furthermorein some biological contextsTETs cooperate with each other to orchestrate specific functions. For instanceTET1 and TET2 are involved in pluripotent reprogramming and imprint erasure induced by cell fusion,142 erasure of 5mC in mouse primordial germ cells,143 pre-mRNA alternative splicing,144 maintaining stem cell identity,145 reprogramming to recover youthful DNA methylation patterns in aged mice146 and epigenetic reprogramming in offspring caused by maternal exercise.147 Binding proteins are required for desired functions in some cases. For exampleupon TGF-β and IL-2 signalingTET1 and TET2recruited by SMAD3 and STAT5bound to and subsequently demethylated FOXP3 promoter to maintain immune homeostasis.148 Similarlyto main bone homeostasisboth TET1 and TET2 were required for demethylating promoters of P2RX7.149 AdditionallyTET1 and TET3 are associated with cerebellar circuit formation150 and CD4 expression in peripheral T cells.151

TET2 and TET3 are required for Treg cell stability and immune homeostasis,152 and improve Treg cell efficacy by increasing the stability of FOXP3.153 TET2 and TET3 acted as recruiters of HDACs to suppress CD86 and prevent autoimmunity.154 Findings also reveal the roles of TET2 and TET3 in embryonic heart development155 and in regulating proper development and maturation of invariant natural killer T cells.156 Knockdown of TET2 led to hyper-proliferation of erythroid progenitorswhereas knockdown of TET3 impaired terminal erythroid differentiation. These findings revealed distinct roles of TET2 and TET3 in the regulation of human erythropoiesis.157 Furthermorethe deletion of TET2 and TET3 led to aggressive myeloid cancer in mice.158 Mice with TET2 and TET3 double knockout in mature B cells developed B cell lymphomawhich can be delayed upon DNMT1 deletion,159 suggesting the importance of proper methylome.

TET1TET2and TET3 are required for somatic cell reprogramming of fibroblasts to pluripotency,160 telomere homeostasis,161 and early body plan formation.162 Human embryonic stem cells (hESCs) with triple-knockout of TET1TET2and TET3 exhibited prominent bivalent promoter hypermethylationsuggesting the role of TETs in maintaining hypomethylation at bivalent promoters to ensure proper lineage-specific transcription during differentiation.163 In mESCsTETs tended to increase demethylation rates at enhancer elements.164 Distinct roles of TETs in regulating 5hmC formationDNA demethylationand gene expression are also explored in cancer cells.165

The overlapping roles of TETs have been explored due to their similar enzymatic activity. Mice with loss of either TET172 or TET2166 are viablewhile most TET1/2 double knockout mice die perinatally,167 suggesting that deletion of the individual TET gene can be compensated by other TETs. InterestinglyTET3 knockout leads to neonatal lethality,128 indicating the unique role of TET3 that could not be compensated by the other TETs. Thusthe overlapping roles of TETs in certain contexts have not yet been fully established. In additionto understand the TETs functions in vivomouse models with gene constitutive or conditional knockout have been generatedsome of which are summarized in Table 4.

Table 4 Representative mouse models with TETs loss of function
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5hmC

TETs-mediated 5hmC formation appears to be an epigenetic markalthough the physiological significance has not been fully elucidated.168,169,170 The 5hmC acquisition occurred in mouserabbitand bovine zygotes,171 indicating that the mark was conserved in these mammalian species. MBD3required for pluripotency in ESCs,172 preferred to binding 5hmC-containing probes rather than 5mC-containing probes and regulated the expression of genes with 5hmC modifications in ESCs.173 In additionthe acquisition of 5hmC by TET1 in enhancers was associated with enhancer activation,174 implying that 5hmC represented a signal mark rather than an intermediate. The idea was supported by the role of 5hmC in germline reprogramming175 and in drug addiction.176 Interestinglyparticular 5hmC acquisition by cocaine lasted at least one month in mouse nucleus accumbens.176 BesidesTET1-mediated 5hmC deposition was also implicated in osteoarthritis.177

Interestingly5hmC formation is not required for the loss of paternal 5mC in early mouse zygotes,178 further supporting the fascinating and mysterious role of 5hmCnot just the demethylation intermediate. 5hmC modifications have been reported to affect protein binding,179 and consistently5hmC might recruit a chromatin-modifying complex to suppress transcription.180 5hmC formation caused by TET3prevented spurious transcriptionwhich was critical for maintaining transcriptional fidelity in the lung.181

Moonlighting functions of TETs

Overexpression of either TET1 or catalytic-death TET1 impaired long-term memory in micesuggesting the catalytic-independent function of TET1.182 FurthermoreTET1 acted as an epigenetic suppressor of thermogenesis in beige adipocytes largely independent of its catalytic activity. SpecificallyTET1 interacted with HDAC1 to suppress key thermogenic gene transcription by reducing histone acetylation.183 Consistentlycatalytic-independent functions of TET1 in silencing developmental genes by regulating H3K27 modifications,184 supported that TET1 acted as an interaction hub for recruiting different chromatin-modifying complexes in a non-catalytic manner.185 Besidesthe non-catalytic function of TET3 in transcriptional repression of SNRPN by binding to SNRPN promoterwas critical for the maintenance of adult neural stem cell state.186

Apart from the ability of DNA oxygenasestudies also unveiled that TET2 could reduce inflammation by repressing IL-6which is independent of its role in converting DNA 5mC to 5hmC. SpecificallyTET2binding with IκBζrecruited HDAC2 to promote histone deacetylationwhich led to the repression of IL-6 at the transcription level. These findings provided a TET2 enzymatic-independent function in repressing specific gene transcription.187

To explore the enzymatic versus nonenzymatic roles of TET2 in hematopoiesisIto et al. performed a comparative analysis of TET2 catalytic mutant mice and TET2 knockout mice. This study found that mice with non-catalytic TET2 mainly developed myeloid malignancieswhile mice with complete loss of TET2 developed both myeloid and lymphoid disorderssupporting the unique non-catalytic role of TET2 in the hematopoietic stem and progenitor cell homeostasis.188

Interestinglybesides its well-known function in regulating the modification of DNATET2 possessed the activity of oxidating 5mC RNA (m5C) into 5-hydroxymethylcytidine (hm5C). Fu et al. found that the catalytic domain of TET2 could induce the formation of hm5C in HEK293T cells. Considering hm5C accounting for approximately 0.02% of total m5C RNA in tumor samplesthis implied the involvement of TET2 in RNA biology.189 Consistentlya study in Drosophila showed that TET protein was involved in the formation of hm5C.190 This study also mapped the distribution of hm5C and revealed hm5C located in coding sequences of many gene transcripts. Importantlyhm5C favors mRNA translation.190 Howeverthe biofunction of hm5C in mammalian RNA is largely unknown until Shen et al. discovered that TET2 was involved in RNA stability.191 These findings uncovered that TET2depending on its enzymatic activity of mRNA oxidationpromoted pathogen infection-associated myelopoiesis. SpecificallyTET2 mediated oxidation of SOCS3 m5Cwhich led to ADAR1 binding and destabilizing SOCS3 mRNA and consequently repressed SOCS3 expression.191 Meanwhileby the TET2 interactome in mouse ESCsGuallar et al. identified that paraspeckle component 1(PSPC1)an RNA-binding proteincould bind to TET2 and this complex recruited HDAC1/2 for repression of MERVL transcription independent of TET2 catalytic activity. More importantlythis study further found that TET2recruited by PSPC1catalyzed hm5C modification of MERVL RNAsfacilitating the degradation of MERVL transcriptsand thus provided a new paradigm for TET2-mediated post-transcriptional silencing of the specific gene. NotablyPSPC1 and its RNA-binding domains are essential for TET2 function in regulating MERVL by both transcriptional and post-transcriptional mechanisms.192 Interestinglyusing a proteomics approachHuang et al. discovered PSPC1 also bound to TET1 for bivalent gene regulation in formative pluripotency independent of the catalytic activity of TET1.193 AdditionallyTET2 has been shown to function in ESC differentiation by reducing the pluripotency-related mRNA stabilitycaused by TET2-mediated hm5C.194 Notablythis study confirmed that TET2 contained an RNA-binding domainwhich had been identified by a proteomic approach in a previous study.195

In addition to its oxidation of mRNArecentlyHe et al. found that TET2 could convert m5C into hm5C in tRNAsubsequently affecting tRNA fragment levels.196 Meanwhilem5C oxidation in tRNA mediated by TET2 facilitated translation.197 These findings linked TET2-mediated tRNA modification to tRNA processing and mRNA translation,196,197 unveiling novel roles of TET2 in gene regulation at multiple levels. Additionallyfindings revealed that TET1/2 could oxidize T to 5hmU in mESCs.198

In this partthe interaction with various binding proteins stably and transiently mainly affects TETs locationincluding recruiting TETs to specific sitesallowing TETs to recruit other proteinsand stabilizing TETs association with DNA. BesidesTETs are capable of oxidizing both DNA and RNA. Understanding the characteristics of TETs might provide key insights into epigenetic editingsuch as DNA demethylation and mRNA modification. Herewe summarize major discoveries in the history of TETs over time (Fig. 4).

Fig. 4
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The timeline of key discoveries in basic research of TETs

TET function regulators

Numerous studies have identified the factors in regulating TETs functionincluding transcription factorsmicroRNAspost-translational modificationsand small molecules in different levelsas summarized in Fig. 5.

Fig. 5
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Primary factors in positive(red) and negative(blue) regulation of TETs activity and the outmost layer described the involved mechanism. FOXA1,201 Vitamin C,238 CRL4(VprBP),224 P300,222 and AMPK223 enhanced TETs activitywhile Vpr,227 IDAX,116 Calpain 1,228 2HG232 and miR-29b207 decreased TETs activity

Transcriptional level

Pluripotency-associated transcriptional factorssuch as MYC and NANOGregulated TET1 expression in hESCs.199 TET1/2 was regulated by Oct4 and SOX2.200 InterestinglyFOXA1 not only transcriptionally regulated TET1but also interacted with TET1 to mediate DNA demethylation of its targeted enhancers.201 BesidesSTAT3/5 transcriptionally activated TET1 expression in AML202 and P53 positively regulates TET1/2 transcription in mESCs.203 SIN3A increased TET1 and TET2 mRNA expression in human pulmonary arterial smooth muscle cells.95 Transcriptional suppression was also identified in the regulation of TETs. NF-κB-mediated repression of TET1 transcription was uncovered in basal-like breast cancer.204 TET3transcriptionally repressed by the nuclear receptor TLXacted as a tumor suppressor in glioblastoma.205

microRNAs

Using the TCGA databasethe miR-29 family were predicted to regulate DNA demethylation by potentially targeting TET1.206 IndeedmiR-29b directly targeted and repressed TET1 to promote the mesendoderm lineage formation207 and the miR-19b/TET1 axis could be utilized in attenuating osteoarthritis progression.208 Downregulation of TET1 also has been reported by miR-494 in hepatocellular carcinoma tumors209 and by miR-191 in intrahepatic cholangiocarcinoma,210 respectively.

Multiple studies have also demonstrated that microRNAs are involved in downregulating TET2 expression. Biochemistry studies discovered that miRNA-22 could directly bind to TET2 mRNA and negatively regulate TET2 expressionwhich contributed to myelodysplastic syndrome and hematological malignancies.211 miRNA-29a could also downregulate TET2 expression.212 A thorough analysis of TET2-targeting miRNA by a high-throughout 3’UTR screenidentified extensive miRNAs such as miRNA-29b and miRNA-101inhibiting TET2 expression and these miRNAs regulated malignant hematopoiesis.213 Further studies identified that TET2 was under control of miRNA-Let7,214 miRNA-210215 miRNA-144-3p,216 miRNA-142-3p,217 miRNA-26a218 and miRNA-10b-5p.219 BesidesTET3 as a miR-150 target was associated with the generation of non-classical monocytes.220

Post-translational modifications

Post-translational modification is also a key process in regulating TETs functions. Bauer et al. found that phosphorylation and O-GlcNAcylation existed in TET2 protein modification,221 indicating that complex modification modulated TET2 functions in different conditions. IndeedP300-mediated acetylation of conserved lysine residues enhanced TET2 stabilityand increased its ability to target chromatinwhich reduced aberrant DNA methylationand thereby protected against abnormal DNA methylation induced by DNA damage.222 AdditionallyAMP-activated kinase catalyzed the phosphorylation of TET2 at serine 99which increased the stability of TET2. While the phosphorylation of TET2 was inhibited under hyperglycaemic conditions such as diabetesconsequently decreasing TET2 levels.223 Monoubiquitylation of TET2 at lysine 1299 mediated by VprBP facilitated TET2 association to chromatinwhereas mutation of TET2 at 1299 blocked its interaction with VprBP and decreased its association with DNA.224 Interestinglythe K1299-linked monoubiquitylation of TET2 could be removed by USP15decreasing TET2 association to DNA.225 Additionallyphosphorylation of TET3 by CDK5 caused lower binding affinity to histone variant H2A.Z. and contributed to higher level of 5hmC at BRN2 promoter to activate BRN2 expression during neuronal differentiation.226

Protein degradation

Surprisinglybesides the CRL4 E3 ligase mediated TET2 monoubiquitylation promoted TET2 association to chromatinHIV-1 derived Vpr hijacked CRL4and this E3 ligase preferred to catalyze polyubiquitylation of TET2accordingly promoting TET2 degradation to sustain IL-6 expression and facilitate viral replication.227 UnexpectedlyIDAXthe TET2-binding proteinpromoted TET2 degradation in a caspase activation-dependent manner.116 With different proteolytic pathway inhibitorscalpains were identified to be involved in TET2 protein regulation. Specificallycalpain 1 was implicated in the degradation of TET2 in ESCsleading to skewing lineage expression.228

Small molecules

As α-KG is required to maintain the oxygenase activity of TETsit is plausible that 2HGgenerated by the reduction of α-KG catalyzed by IDH enzyme mutants,229 might disrupt TETs function.230 Indeedbiochemistry studies demonstrated that mutant IDH decreased TET2-mediated 5hmC levels.231 Consistentlystructure analysis revealed that 2HG occupied the site of α-KG in protein conformational spacesuggesting that 2HG served as a competitive inhibitor of α-KG-dependent enzyme activityincluding TET2.232 In addition to 2HGsuccinate and fumarate were also identified to act as α-KG antagonistswhich inhibited TET2 dioxygenase activity.233 RecentlyChen et al. found that itaconate was also a TET2 dioxygenase inhibitor through the competition with α-KG to interact with TET2resulting in dampening inflammatory responses.234 Besidesthe nuclear glutamate dehydrogenase interacted with TET3 to supply TET3 with αKG and increased its demethylation activity in neurons.235

Previous studies have revealed that vitamin C could upregulate the activity of some α-KG-dependent dioxygenasessuggesting that vitamin C might be involved in the modulation of TETs activity. Indeedvitamin C could enhance TET2 activity and subsequently increase 5hmC levels in ESCs.236,237 Yin et al. found that vitamin Cbut not other reducing chemicals such as NADPH and vitamin Ewas a unique activator of TET dioxygenases.238 It is possible because vitamin C was capable of binding to the catalytic domain of TET proteinsfacilitating protein foldingand accelerating oxidation reactions.238 The ideathat vitamin C acting as a TET agonistwas reinforced by a series of further studies.239,240,241,242,243,244,245 NotablyTET2 deficiency presented in aberrant self-renewal and leukemia progressionwhich can be blocked by treatment with vitamin Csuggesting that vitamin C treatment might be beneficial to patients with leukemia.246 Specificallyvitamin C restored TETs function and drove the expression of related genes.246

Aside from metabolitesThienpont et al. found that the activity of TET2 was reduced under hypoxic conditionsleading to DNA-hypermethylation.247 Oxygen levels determined the activity of TET1 in ESCs.248 Redox-active quinones promoted the production of 5hmC by TETs.249

Artificial inhibitors and activators of TETs have also been explored. By screening strategya small molecule compoundC35was identified as a TETs inhibitor. Notablythis compound specifically blocked TETs catalytic activities without abolishing TETs complexes.250 Bobcat339one of synthesized cytosine derivativesinhibits TET1 and TET2 activity.251 A small moleculeTETi76inhibits TETs specifically.252 InterestinglyNickel (II) exhibits inhibition to TETs enzymatic activities by replacing the cofactor Fe (II) of TETs.253 AdditionallySRT1720a SIRT1 agonistby deacetylating TET2significantly increases TET2 activity.254

Togethersimilar to other genesTETs can be regulated at multiple levelsincluding post-transcriptional and post-translational regulation. Furthermoreit can be modulated by small molecules involved in its enzymatic reaction. This ensured the fine-tuning of TETs enzymatic activity in response to external cues.

Targeted therapy and clinical trials

Given the various roles of TETs in biological processesit comes as no surprise that it has been proposed as an important therapeutic target for diseases such as cancer.113,114,252,255,256 For examplevitamin Cby improving TETs activityallows leukemia cells to be more sensitive to PARP inhibitors.246 Interestinglycells with TET2 mutationspossibly heavily relying on compensatory roles of TET1/3showed more vulnerable to TETs inhibitors compared with normal ones. These findings provide a new therapeutic strategy for selective targeting of cells bearing TET2 mutations.252 5-azacytidinea DNA demethylating agentshows higher cytotoxicity in TET2-silenced cellsprobably due to the hypermethylation pattern caused by the loss of TET2.256 In additionC35a selective TETs inhibitorpromotes somatic cell reprogramming.250 As a robust TET2 activatorclinical trials are investigating the effects of Vitamin C on hematologic malignancy patients with TET2 mutations (NCT03397173; NCT03433781). Of notethe antitumor effects of vitamin C has been studied for a long time; howeverits efficacy against cancers have not been established by clinical trialspossibly because of the complex mechanisms of action of vitamin C.257,258,259,260,261,262 As a new targetthe role of TET2 enzymatic activity enhanced by vitamin C in patients with hematological malignancies remains unclear. Besidesclinical trials evaluating the contribution of vitamin C-mediated upregulating TET2 enzymatic activity in solid tumors are urgently required. Notablyhigh concentrations of vitamin C administration with or without anticancer drugs have not shown serious adverse effects in clinical trialssuggesting that vitamin C is a drug with low toxicity.263,264,265,266,267 Thereforevitamin C might be a promising anticancer treatment option for cancer patients with dysfunctions of TET2 in the future.

Detection of 5hmC

5hmC plays distinct epigenetic roles in mESCs.268,269 In additionaberrant levels of 5hmC are associated with various cancers.270,271,272,273,274,275,276,277,278 Furthermore5hmC signatures in circulating cell-free DNA can be used as biomarkers for cancer diagnosis.279,280,281,282,283 Togethermapping the distribution of 5hmC in a genome is important not only to elucidate its biologysuch as functions in developmentbut also to use it for clinical potential.284,285,286,287,288 In this sectionrepresentative approaches for detecting 5hmC with or without bisulfite treatment are discussed (Fig. 6).

Fig. 6
Fig. 6
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Representative schematic diagrams of 5hmC detection approaches. a Schematic diagrams of hMe-Seal,296 TAB-seq,297 oxBS-seq,298 hmC-CATCH,299 CAPS,301 Jump-Seq,302 ACE-Seq,303 and CAM-Seq.304 b Structural formula of representative molecules

hMeDIP

To investigate the global distribution of 5hmCanti-5hmC antibodies were utilized to capture 5hmC DNA from genomic DNA followed by sequencingand this approach was named as hMeDIP.289,290,291 This method is cost-effective and widely used. Howeverthe biggest limitation of this method is the quality of anti-5hmC antibodies. To solve the problem caused by using antibodies of different production batchesRobertson et al. developed a novel 5hmC detection methodbased on the selective glycosylation of 5hmC treated with β-glucosyltransferase. This β-glucosyl-5-hydroxymethylcytosine-containing DNA could be efficiently and specifically captured by J-binding protein 1. After enriching 5hmCfurther analysis could be performedsuch as qPCR and sequencing.292,293 Likewise5hmC was converted to cytosine-5-methylenesulfonate (CMS) upon sodium bisulfite treatmentand then the CMS-specific antiserum was used to capture CMS-containing DNA fragments for further analysis.294,295

hMe-Seal

Bisulfite treatment could lead to significant degradation of DNAand therefore bisulfite-free methods were developed for limited DNA samples. For exampleβ-glucosyltransferase could convert 5hmC to β-glucosyl-5-hydroxymethylcytosine (5gmC) in the presence of UDP-Glu. The 5hmC can be labeled with an azide group using the modified UDP-Glu with the azide. This allowed biotin moiety containing an alkynyl group to link to 5hmC using click chemistryfollowed by affinity enrichment and sequencing.296

TAB-Seq

In 2012Yu et al. developed a TET-assisted bisulfite sequencing approachnamed as TAB-Seqwhich enabled the detection of genomic 5hmC sites at single-base resolution. Specifically5mC could be oxidized to 5caC with TET proteins and the 5caC could subsequently be deaminated to form U by bisulfite treatmentwhile the glucosylated-5hmC was protected from TET oxidation and bisulfite deamination and therefore was identified as C. This method allowed discriminating 5hmC from 5mCin contrast with traditional bisulfite sequencing.297

oxBS-Seq

MeanwhileBooth et al. also developed a method of quantitatively mapping 5hmC distribution at single-base resolutionknown as oxidative bisulfite sequencing (oxBS-Seq). This approach utilized potassium perruthenate to selectively oxidate 5hmC to 5fC that was subsequently converted to U by bisulfite treatmentwhile 5mC was not oxidized by potassium perruthenate and still detected as C. This method enabled the determination of the amount of specific 5hmC sites by subtracting the readout of traditional bisulfite sequencing.298

hmC-CATCH

Similar to oxBS-Seqpotassium ruthenate was used to convert 5hmC to 5fCwhich was further selectively modified with an azidoand this adduct was identified as T during PCR. Thereforethe C-to-T transition was regarded as the readout of 5hmC. Additionallythe azido group rendered it easily for enrichment and sequencing.299

CAPS

Similar to TAB-seqTETs were employed to convert both 5mC and 5hmC to 5caCand pyridine borane was subsequently used to convert 5caC to dihydrouracilthat was read as T during PCR. This modified C-to-T transition allowed whole-genome detection of 5mC and 5hmC at single base-level resolution. In contrastglucosylated-5hmC was inert to TET oxidation and borane reductionand thus 5mC sites could be analyzed specifically.300 Accordinglythe amount of 5hmC sites could also be determined by comparing the readouts with or without β-glucosyltransferase treatment at the first step. AlternativelyTET proteins could be replaced by potassium perruthenate to selectively oxidate 5hmC to 5fCallowing specifical sequencing of 5hmC.301

Jump-seq

A new strategycalled Jump-seqwas developed by Hu et al. for detecting 5hmC without sequencing the whole genome at nearly a single-base resolution. This method took advantage of selectively labeling 5hmC with a glucose moiety carrying an azide groupfollowed by linking a hairpin DNA with an alkyne group. 5hmC positions could be deduced by the connection between genomic DNA sequence and the hairpin sequence after primer extension.302

ACE-seq

APOBEC3A-based 5hmC sequencing methodnamed ACE-seqhas been developed without bisulfite treatment at single-base resolution. 5hmC was modified with glucose by β-glucosyltransferase and the glucose-modified 5hmC was inert to APOBEC3Aa DNA deaminasewhereas C and 5mC could be converted to Uyielding 5hmC identified particularly.303

CAM-Seq

With a similar strategy5hmC was initially converted to 5fC by KRuO4. Then using azi-BPa compound reported by the same group5fC was selectively labeledrendering it matching with A and identified as T by PCR. Using this method 5hmC loci in genomic DNA could be analyzed at single-base resolution.304

As the findings of the important role of TET families in DNA modificationselective chemical labeling of the hydroxyl group of 5hmC is fast-growing to map the genome-wide distribution of 5hmC. Herewe summarize some characteristics of each method in Table 5. Of noterecentlynanopore sequencing technologies have shown a diverse range of applicationsincluding 5hmC detection.305,306 In additionhm5C could be detected by mass spectrometry.307,308 Like hMeDIPhm5C-containing RNA could be captured by the anti-hm5C antibody followed by sequencing and this method was named as hMeRIP-seq.190,194

Table 5 Representative approaches for 5hmC detection
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Demethylation editing tools

Dynamic regulation of DNA methylation and demethylation plays a critical role in many biological processesincluding epigenetic memorygenomic imprintingand development.309,310,311 Dysregulation of this process leads to many diseases such as autoimmune disorders and cancers.312,313,314 In additionhypermethylation patterns are usually associated with gene silencing. Thereforedeveloping epigenetic editing tools allow us not only to modify the target locus to evaluate the consequences of epigenetic marksbut also to silence or activate the gene in specific contexts. The general idea of epigenetic editing is that an epigenetic writer or eraser is fused to a sequence-specific DNA-binding domain to rewrite the epigenetic marks in targeted loci or histone315,316(Fig. 7). In this partwe summarize TETs-based epigenetic editing tools (Table 6).

Fig. 7
Fig. 7
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Representative working models of targeted demethylation. a With the gDNAa DNA modification domainsuch as TET1fused to dCas9led to the erasure of specific DNA methylation.333 b GCN4 repeats fused to dCas9recruited many copies of an anti-GCN4 antibody (scFV)-fused TET1to amplify demethylation efficiently.322 c Multiple effectors were used to increase the efficacy of demethylation. The modified gRNA with PUF binding sitesrecruited protein fusions of PUFTET1and NEIL2 to particular DNA methylation sites. Among thesePUF were used for binding to the modified gRNAand TET1 oxidated 5mC and NEIL2 worked as a DNA glycosylase to promote DNA demethylation.324 d Without tethering an effector such as TET1only gRNA-dCas9 led to specific DNA demethylationby sterically blocking DNA methyltransferase331

Table 6 Representative demethylation tools
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TET1-TALE-fused-based tools were developed for epigenetic editing,317,318 and successfully increased β cell replicationdemonstrating a promising approach in therapeutic applications.318 Customized TALE repeat arrays worked as a platform for guiding TET1 to the DNA sequence of interesttherefore leading to the demethylation of targeted lociand subsequently increasing the related gene expression.317

Additionallyengineered endonuclease-dead Cas9 (dCas9) could also be used as a linkerand recruited indirectly or fused directly to the designed effector domainssuch as TET1to modify the specific target in conjunction with gRNA.319,320,321,322,323 Furthermoreco-delivery of demethylation pathway-related proteins such as GADD45A and NEIL2with dCas9-TET1enhanced demethylation editing efficacy.324 Besidesthe CRISPR/dCas9-based gene transcription activation system coupled with TET1activated silenced genes through demethylating.325,326

In addition to dCas9other DNA-binding domains worked as a target loci modification guider. For examplea synthetic fusion proteincarrying enzymatic domains of TET1 and reverse tetracycline transactivatorexhibited demethylation of Tet promoterupon doxycycline treatment.327 SimilarlyTET2 was fused to a DNA-binding domain to promote the demethylation of targeted lociand thereby a TET2-based editing approach was developed.328 The engineered protein contained two core domains: TET2 for inducing DNA demethylation and zinc fingers for binding the ICAM-1 promoters.328

Other effectors could also be employed such as TET3 and ROS1. TET3 catalytic domainsfused to dCas9could produce 5hmC formation.329 Plants DNA demethylases such as ROS1 could replace TET1 to induce demethylation.330 Interestinglysimple CRISPR/dCas9 and gDNA without tethering any other enzymes appeared to demethylate target loci efficiently largely due to steric blockage of DNA methyltransferase.331

Methylation editing tools have shown great potential in clinical research and treatment. Model mice with Silver-Russell syndrome has been successfully generated by TET1-dCas9 based system.332 TET1-based DNA methylation editing could restore the expression of FMR1 by demethylating its promotersupporting the potential application of epigenome editing in fragile X syndrome treatment.333 SimilarlyTET1-dCas9 mediated demethylation of the MECP2 promoterrescued Rett syndrome neurons.334 Thusprecise and efficient epigenetic editing tools would provide new insights into the functions of the specific DNA modification locus temporal-spatially.

Summary

Here we review the remarkable findings in understanding the function of TETs in modifications of DNA and RNAand summarize recent advances in the detection of 5hmC and DNA demethylation editing tools. Despite the formation of oxidation products (5hmC5fCand 5caC) and the mechanism of active DNA demethylation have been characterizedsome questions have yet to be answered. Firstthe significance of 5hmC needs to be delineated. Secondin addition to oxidating DNArecent studies have also demonstrated that TET2 is capable of oxidating RNA. It is still not well-defined what factors determine TET2 in choosing oxidating DNA or RNA. Thirdregardless of containing DNA-binding domainall TETs appear to be recruited to specific DNA sequences by their binding partners. It is worth to further explore how to modulate the binding of TETs to its target DNA sequences in various biological processes. Fourthloss of function mutations of TET2 are frequently identified in blood malignancieswhereas mutations of TET2 are uncommon in solid tumors. Howeversignificant downregulation of TET2 activity is observed in many solid tumors. The underlying mechanisms are still not clear and require to be explored for the diagnosis and therapy of cancers. We believe that addressing the questions above will help us further understand the roles of TETs in the occurrence and development of many diseases.