TET enzymes, DNA and pluripotency

Samuel E Ross1, 2 and Ozren Bogdanovic1, 3

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

Abstract Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3) actively cause demethylation of 5-methylcytosine (5mC) and produce and safeguard hypomethylation at key regulatory regions across the . This 5mC erasure is particularly important in pluripotent embryonic stem cells (ESCs) as they need to maintain self-renewal capabilities while retaining the potential to generate different cell types with diverse 5mC patterns. In this Review, we discuss the multiple roles of TET proteins in mouse ESCs, and other vertebrate model systems, with a particular focus on TET functions in pluripotency, differentiation, and developmental DNA methylome . Furthermore, we elaborate on the recently described non-catalytic roles of TET proteins in diverse biological contexts. Overall, TET proteins are multifunctional regulators that through both their catalytic and non-catalytic roles carry out myriad functions linked to early developmental processes.

Introduction Ten-eleven translocation (TET) methylcytosine dioxygenases were first described when TET1 was identified as a fusion partner of the mixed lineage leukaemia gene (MLL) in acute myeloid leukaemia [1]. Since then TET proteins have been associated with other myeloid and lymphoid malignancies as well as solid cancers including melanoma, breast, and prostate cancers [2]. TET proteins play key roles in the regulation of self-renewal capacities of diverse stem cell types, and mutations in genes coding for TET proteins can lead to oncogenic transformation [3]. The major catalytic function of TET proteins was first described in a landmark study, which revealed that TET1 could catalyse the conversion of 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC) [4]. It is now known that TET proteins can cause the sequential oxidation of 5mC to 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) [5, 6]. The oxidised intermediates, 5fC and 5caC, can be removed by thymine DNA glycosylases (TDG) and base-excision repair (BER) machinery to regenerate unmethylated at targeted sites [7, 8]. Specific reader proteins have been described for each of these oxidised intermediates, which is suggestive of their potential function in gene regulation [9]. This ability of targeted 5mC removal associated with TET function is particularly important in embryonic stem cells (ESCs) as they need to maintain self-renewal capabilities as well as adopt diverse 5mC patterns upon differentiation. Mammals and vertebrates such as zebrafish encode three TET protein copies (TET1, TET2, TET3) [4, 10] whereas in the Xenopus genus only TET2 and TET3 have been described [11]. Invertebrate chordates such as amphioxus (Branchiostoma lanceolatum) display a single TET orthologue of yet uncharacterised function [12]. TET proteins are characterised by their core catalytic domain and can exist in cell-type specific isoforms with or without their CXXC binding domain (present in TET1 and TET3, and supplemented in TET2 by its association with CXXC4/IDAX) [13, 14] (Figure 1). In this Review, we focus on insights related to TET protein function in mouse embryonic stem cells (mESCs) and vertebrate embryos. More extensive reviews of TET function have recently been published elsewhere [2, 15].

TET expression and binding dynamics in mESCs ESCs are derived from epiblasts of mammalian blastocysts and cultured in conditions that promote propagation of the pluripotent state. ESCs are considered to be either ‘naïve’ when isolated from pre-implantation epiblasts (E3.5-E4.5), or ‘primed’ when obtained from post- implantation epiblasts (E5.5-E6.5) [16, 17]. Naïve mouse ESCs are generally cultured in two types of media. The original culture conditions consisted of leukaemia inhibitory factor (LIF)

and foetal calf serum. While these conditions efficiently maintained the potential for blastocyst chimera formation, cells in these cultures displayed heterogeneous expression patterns of pluripotency genes. Furthermore, such cells exhibited 5mC patterns similar to post- implantation embryos and somatic cells [17]. More recently, culture conditions consisting of serum-free media and two small molecular inhibitors (2i) that block FGF/ERK pathway and partly inhibit glycogen synthase 3 (Gsk3), have been developed [18]. Cells grown in 2i conditions are homogeneous in terms of pluripotency marker expression and morphology, in addition to displaying low 5mC levels characteristic of pre-implantation embryos [19-21]. Such cells are thus naïve both in terms of their cellular potency and their epigenome, and are often referred to as “ground state” naïve ESCs. The majority of earlier studies (e.g. before 2010) routinely used serum/LIF conditions for mESC culture, thus unless otherwise stated, the studies discussed below refer to serum/LIF-cultured mESCs. In mESCs, Tet1 and Tet2 constitute the majority of Tet transcripts while Tet3 is barely detectable [5, 22, 23] (Figure 2). When LIF is removed from the mESC culture media, mESCs spontaneously differentiate and this is marked by a significant decrease in Tet1 and 5hmC levels [4, 22]. In line with these findings, Tet1 levels displayed a progressive drop during the eight days of embryoid body (EB) culture [22]. This is in contrast to Tet2 transcripts that decrease during first four days but are fully restored by day eight, and Tet3 transcripts that displayed a progressive and significant (> 20 fold) increase during this process [22] (Figure 2). It is thus likely that the relatively high 5hmC content (~ 4%) observed in undifferentiated ESCs [4] is predominantly caused by high levels of TET1, and to a lesser extent, TET2. In agreement with these findings, in 2i-cultured ESCs, Tet1 is the most highly expressed Tet transcript whereas Tet3 is undetectable [19]. Interestingly, 2i and serum/LIF conditions differ in the levels of Tet2, which appears to be more highly expressed in 2i [19]. During serum to 2i reprogramming, TET1 and TET2 are required for the production of 5hmC even though they are not the major determinants of the profound 5mC loss associated with this process [19, 20]. Both serum to 2i reprogramming as well as the reprogramming of epiblast stem cells (EpiSCs) to naïve pluripotency can be enhanced by the addition of retinol and ascorbate to the growth medium [24]. These components augment TET activity by different mechanisms; ascorbate directly stimulates TET activity by the reduction of non-enzyme bound Fe3+ to Fe2+, whereas retinol and retinoid acid (RA) increase expression of Tet2 and Tet3 through binding to conserved RA-responsive elements [24]. Genome wide profiling of 5hmC and TET1 protein binding sites have revealed an enrichment of TET1 at CpG island promoters and gene bodies in mESCs, in agreement with

the presence of the CXXC domain in TET1 [25-27] (Figure 1). Nevertheless, the exact role of CXXC in TET1 targeting to chromatin is unclear, and it appears that TET1 largely depends on factors such as NANOG, PRDM14, TEX10, and others for its chromatin recruitment [28-30]. TET1 also exhibits strong enrichment at bivalent promoters that are marked by both active (H3K4me3) and repressive (H3K27me3) histone marks [27, 31]. These regulatory signatures frequently decorate promoters of developmental and pluripotency genes [32]. Similarly to TET1, TET2 can also associate with gene bodies [33] and distal regulatory elements such as enhancers [34]. The exact roles of TET protein function in mESCs are far from being fully understood. So far, TETs have been attributed both activator [25] and repressor [26] function, and have been associated with the regulation of bivalent chromatin [31] and Polycomb-marked regions [27]. These functions, when impaired, are believed to impact diverse features of the pluripotent transcriptome with profound consequences for differentiation processes [23, 34] (Table 1).

TET proteins play key roles during mESC differentiation Despite the coordinated expression of TETs during pluripotency, TET triple knockout (TKO) mESCs maintained self-renewal capabilities, normal ESC morphology, and expressed pluripotency markers such as Oct4 and Nanog [35, 36]. However, embryoid bodies (EBs) formed from TKO mESCs displayed reduced levels of endo- and mesodermal markers and resulted in poorly differentiated tissues in general [35]. Moreover, TKO teratomas lacked mesodermal and endodermal structures whereas TKO mESCs were characterised by low efficiency of chimeric embryo formation [35]. A number of studies have explored the effects of single and double TET knockouts (DKO) to reveal specialised roles of TET proteins during ESC differentiation (Table 1). TET1 targeting studies have reproducibly found that TET1 depletion in mESCs results in a skew towards extraembryonic, mesodermal or endodermal lineages in embryoid body differentiation assays and teratoma formation assays [5, 23, 25, 36- 38] (Figure 3A). This can be partly explained by the observation that TET1 associates with the transcriptional repressor and activator SIN3A to activate the nodal antagonist Lefty1 in mESCs [39]. Depletion of TET1 results in Lefty1 promoter hypermethylation and decreased expression of Lefty1 and Lefty2 transcripts as well as in an increase in mesoderm/endoderm transcription factors T and Foxa2 [23, 37, 39]. Moreover, TET1 depletion causes an increase in trophectoderm markers such as Cdx2, Eomes, and Hand1, and a decrease in neuroectoderm markers like Pax6 [23] (Figure 3A). The preference toward mesodermal and endodermal lineage could also be a result of decreased expression of pluripotency markers associated with

restricting endoderm formation such as Esrrb and Prdm14 [25]. TET1 depletion in mESCs also leads to bivalent promoter DNA hypermethylation followed by an unexpected increase in the accompanying levels [27, 36, 37]. It is hypothesised that TET1 at bivalent promoters recruits Polycomb Repressive Complex 2 (PRC2) to suppress gene expression and that hypermethylation of these loci would inhibit PRC2 binding. This is supported by observed physical interactions between TET1 and PRC2 and overlaps in PRC2, TET1 and 5hmC genomic profiles in mESCs [40]. Unlike TET1, TET2 depletion in mESCs has no visible effect on the differentiation outcomes of mESCs in embryoid body and teratoma formation assays [23, 34]. TET2 depletion, however, results in delayed expression of genes associated with early differentiation stages and also causes a significant reduction in cellular 5hmC levels [34]. As TET2 mainly associates with enhancers, many of which exhibit low occupancy, its role in mESCs appears to be the priming of regulatory regions for activation upon differentiation [33, 34]. Unlike Tet1 and Tet2, Tet3 is expressed at low levels in mESCs [5, 22, 23]. TET3 only contributes to 2% of the total 5hmC in mESCs as assayed by mass spectrometry [36]. However, knockout of Tet3 in mESCs causes impaired neuroectoderm formation in serum-free embryoid body assays [41]. This observed skewing is likely caused by the lack of Wnt signalling suppression, driven partly by promoter hypermethylation and decreased expression of Wnt inhibitor secreted frizzled-related protein 4 (Sfrp4) upon Tet3 depletion [41]. Additionally, studies in induced pluripotent stem cells (iPSCs) have also supported the notion of TET proteins as regulators of differentiation. Tet1 alone can be used as a substitute for Oct4 in the renown OKSM (Oct4, Klf4, Sox2, c-myc ) reprogramming cocktail [42], and TET proteins have proven essential for the reprogramming of multiple somatic cell types such as mouse embryonic fibroblasts (MEFs), neural cells, and B cells [28, 43, 44]. Unexpectedly, however, TET proteins only promote reprogramming in the absence of vitamin C despite it being known to increase TET activity [45, 46]. In summary, TET TKO and single TET KO studies reveal that while TET proteins are not required for mESC pluripotency, they are essential for the maintenance of proper differentiation capacity and the generation of functional embryonic structures. Notably, TET proteins seem to participate in the regulation of genes with well-established roles in the suppression of developmental pathways. TET proteins and developmental DNA methylome reprogramming While DNA methylomes are generally stable in mammalian somatic cells, the mammalian life cycle is characterised by two global 5mC reprogramming events [47, 48]. The first genome-

wide 5mC erasure takes place shortly after and is characterised by the rapid removal of 5mC from the paternal pronucleus [49, 50], followed by a progressive drop in 5mC levels associated with both maternal and paternal genomic contributions [51]. 5mC levels reach their lowest point during the blastocyst stage, after which the methylomes are gradually re- established during gastrulation. Similarly, during primordial germ cell (PGC) formation, 5mC is globally erased and re-established for the second time, and this event is thought to be crucial for sex-specific imprint establishment [52, 53]. Given the roles of TET proteins in DNA demethylation, a number of studies have interrogated the links between TETs and global 5mC erasure in mammals. Initial studies have established links between TET3-mediated DNA and the reprogramming of the zygotic paternal DNA methylome, following fertilisation [54]. However, more recent work suggests that inhibition of TET3 activity, as well as Tet3 deletion, reduces the amount of zygotic 5hmC, without affecting paternal 5mC erasure [55]. This implies that TET3 plays a protective role in safeguarding hypomethylated genomic sites rather than being the initiator of this major demethylation event. Similarly, PGC-specific DNA demethylation can occur in the absence of TET1 and TET2 activity in vitro [56] and in vivo [57, 58]. While TET1 loss resulted in ~50% reduction of global PGC 5hmC, the TET1 KO PGC genome reached a hypomethylated state at E13.5 comparable to its wild type counterpart [58]. It is worth noting, however, that Tet1-deficient embryos display abnormalities associated with imprint erasure [59]. In summary, TET proteins are important regulators of the genomic 5hmC content, yet their contribution to mammalian global DNA demethylation events is likely only auxiliary.

Gastrulation and body plan formation in vertebrates is TET-dependent To assess the roles of TET proteins in vivo, diverse knockout and knockdown strategies have been employed in mouse, zebrafish and Xenopus (Table 1). Adult -specific deletion of all three TET proteins in mice followed by crossing resulted in TKO progeny that were still able to form all three embryonic germ layers [60 ]. However, these TET TKO embryos did not develop past gastrulation and had no discernible early organ structures [60]. These mice also displayed diffused expression of extraembryonic markers like Eomes and downregulation of nodal agonists Lefty1 and Lefty2 [60]. This was followed by another mouse TKO study that demonstrated hyperactive Wnt signalling in neuromesodermal progenitors at similar embryonic stages (E7.5), suggestive of both Wnt and Nodal signalling being involved in TET- dependent regulation of embryonic patterning [41]. Whereas TET TKOs are characterised by lethal gastrulation defects, phenotypes observed upon deletion of single TET proteins are less

severe and their importance for survival are varied. TET2 KO mice develop normally but are prone to myeloid malignancies and this in agreement with the absence of phenotypes observed upon TET2 KO in mESCs [61, 62]. Maternal deletion of Tet3 resulted in normal preimplantation development, however the affected embryos displayed morphological abnormalities starting from midgestation, and a severely reduced viability rate [54]. Tet1/3 double knockouts (DKOs) were embryonically lethal and no viable embryos could be detected after E10.5 [63]. The phenotypes of single Tet1 knockouts appear to depend on the genetic background. Tet1 KOs on a mixed genetic background are viable [37], whereas C57BL/6 KO Tet1 KO mice display partial embryonic lethality [63, 64]. The majority of the defects observed in Tet1 KO were associated with gastrulation and involved impairment of primitive streak formation and misregulation of metabolic genes in the extraembryonic ectoderm [64]. Finally, the combined deficiency of TET1 and TET2 causes severe developmental abnormalities including exencephaly and growth retardation and is perinatally lethal; the majority of Tet1/Tet2 DKO mice die within two days after birth [65]. Interestingly, a fraction of Tet1/Tet2 DKO mice can survive to adulthood with only minor abnormalities. These mice displayed imprinting defects and somewhat higher levels of global 5mC as measured by MeDIP-seq approaches. Furthermore, these mice exhibited increased levels of Tet3, which is believed to compensate for Tet1/Tet2 loss [65]. In zebrafish, tet expression starts between late gastrula and early somitogenesis stages and coincides with a global increase in 5hmC abundance [10]. tet1/2/3 TKO zebrafish generated using TALE technology develop past the gastrulation stage and die in the larval period displaying eye, brain, and Notch signalling defects [66]. Another study, which used morpholino knockdown (MO) approaches to deplete tet1/2/3 in zebrafish, demonstrated that affected embryos do not pass gastrulation [67], as previously shown in TET TKO mice [60]. These tet1/2/3 MO embryos also displayed specific DNA hypermethylation of conserved developmental enhancers related to genes involved in TGF-β, Notch/Delta, and Wnt signalling pathways [67]. Furthermore, this TET-dependent demethylation of embryonic enhancers is a conserved feature of the vertebrate phylotypic stage, the most conserved period of vertebrate embryogenesis [67]. In the frog Xenopus laevis, tet3 MO depletion results in severe eye and neural phenotypes and greatly reduces the expression of neural development genes like pax6 [11]. These phenotypes are in agreement with phenotypes observed upon zebrafish tet3 MO knockdown that was characterised by microphthalmia [67]. In summary, vertebrates display diverse requirements for TET proteins during embryogenesis with specific TET proteins playing both common and organism-specific roles within the vertebrate subphylum. A major

feature, consistent across all vertebrates, is the lack of requirement for TET proteins during pluripotency, and the absolute requirement for TET function during gastrulation and body plan formation (Figure 3B). This requirement is underpinned by the timely activation of distal regulatory regions by means of active DNA demethylation, associated with key developmental pathways.

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DNA Demethylation-Independent Functions of TETs While the major described function of TET proteins is the sequential 5mC oxidation, a growing body of work has now identified important non-catalytic functions for TETs in diverse model systems. A recent study using full-length TET1 knockout mice found that their phenotypes differed from other catalytic TET1 KO mice and therefore proposed an important non-catalytic role for the N-terminus of TET1 in embryo development [64]. TET2 can also regulate gene expression by directly interacting with O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) to promote histone O-GlcNAcylation, which is a process that appears to be independent of TET2 enzymatic activity [68]. This can subsequently promote H3K4me3 deposition through the SET1/COMPASS complex [69]. Notably, this interaction with OGT has also been proposed to enhance and regulate the catalytic activity of TET1 as demonstrated in a recent study that utilised zebrafish embryos and mESCs [70]. TET proteins may also possess catalytic activity towards RNA and not just DNA. Recently it has been shown that Paraspeckle Component 1 (PSPC1), an RNA binding protein, directly binds TET2 and directs it to MERVL transcripts where TET2 then demethylates and destabilizes the RNA leading to a decrease in its expression [71].

Conclusions and perspectives

• TET depletion studies in mESCs and other vertebrate model systems have revealed both specialised and redundant roles for TET proteins in differentiation, gastrulation, and body plan formation but not during pluripotency; TETs appear to be dispensable for pluripotency both in vivo and in vitro [35, 60, 67]. The roles of TET proteins are currently under intense investigation because of their apparent links with cancer [2] and clonal hematopoiesis [72]. • Many questions related to TET function remain unanswered. For example, no unifying mechanism that describes TET targeting to chromatin has been revealed to date.

Biochemical studies have identified a number of transcription factors that are proposed to aid TET recruitment to DNA [28-30, 44], however it is not yet clear how evolutionarily conserved these mechanisms are. Moreover, the exact role of 5hmC in gene regulation is far from being understood. TKO studies in mice and tet1/2/3 morphants in zebrafish suggest that TET activity is required for demethylation and activation of distal regulatory elements associated with developmental pathways [35, 41, 60, 66, 67]. It has been proposed that such 5mC to 5hmC conversion could participate in the reconfiguration of chromatin structure that might be required for subsequent transcription factor binding [73]. This is supported by notions that hmC- marked nucleosomes are less stable in vitro [74] and that the methyl-CpG-binding domain (MBD) of a key 5mC-dependent repressor, MeCP2, binds 5hmC ~20 fold less stronger than 5mC [75]. • With the advent of single cell epigenome profiling technologies [76] TET proteins and 5hmC are again gaining significant traction. A recent study demonstrated the utility of single-cell 5hmC sequencing in lineage reconstruction during early mammalian embryogenesis [77]. It is expected that such high-resolution epigenome profiling techniques will greatly aid in disentangling the complex relationships between 5mC, 5hmC, and genome regulation, during vertebrate and disease formation.

Funding Australian Research Council (ARC) Discovery Project (DP190103852) supported this work. O.B. is supported by NHRMC (R.D. Wright Biomedical CDF APP1162993) and CINSW (Career Development Fellowship CDF181229).

Competing Interests The Authors declare that there are no competing interests associated with the manuscript

Table 1. Overview of TET depletion studies in mESCs and vertebrate embryos

Model Depletion strategy Phenotype Reference

mESC Tet1, Tet2, Tet3 Impaired mESC self-renewal and maintenance, and [5] shRNA KD morphological abnormalities upon Tet1 KD

Tet1, Tet2, Tet3 Global loss of 5hmC, increase in trophectoderm (Cdx2, [23] siRNA KD; Tet1, Eomes, Hand1), decrease in neuroectoderm (Pax6 and Tet2 shRNA KD Neurod1), and decreased Nodal antagonist (Lefty1 and Lefty2) expression in Tet1/Tet2 KD. Minor increase in Pax6, Neurod1, Lefty1/2 expression upon Tet2 KD. Tet3 depletion causes Lefty2 repression.

Tet1/2 siRNA KD; Global loss of 5hmC, down-regulation of pluripotency [25] Tet1 shRNA KD genes, increased expression of extraembryonic endoderm differentiation markers, and increased Cdx2 expression upon Tet1/Tet2 knockdown

Tet1 KO Altered expression of lineage specification markers T and [37] Pax6, low efficiency of EB formation, loss of 5hmC and trophectoderm-like cells in Tet KO teratomas

Tet1 siRNA KD Loss of stem cell identity, morphological changes, global [38] 5hmC loss, impaired LIF/Stat3 signalling, and upregulation of differentiation markers

Tet1/2 DKO Global reduction of 5hmC levels, trophectdoerm-like cells [65] in Tet1/2 DKO teratomas

Tet1/2/3 TKO Global loss of 5hmC , defects in EB and teratoma [35] formation, poor contribution to chimeric embryos, increased promoter 5mC, deregulation of developmental genes

Tet1 TKO; Tet2 KO hmC loss of 44% and 90,7% in Tet1 KO and Tet2 KO [34] respectively, increased 5mC on enhancers and delayed gene induction during ES-NPC differentiation. in Tet2 KO,

Tet1/2/3 TKO Lower (< 5%) global 5mC levels, increased 2C-like [36] population in Tet TKO ESCs

Tet3 KO; Tet1/2/3 Skewed differentiation toward cardiac mesoderm and [41] TKO down-regulation of Wnt signalling inhibitor Sfrp4 during differentiation of Tet3 KO mESCs, decreased neuroectdoerm formation in Tet1/2/3 TKO mouse Tet1 KO 75% of homozygous pups displaying smaller body size [37]

Conditional Tet2 Enlargement of the hematopoietic stem cell compartment, [61] KO (hematopoietic splenomegaly, monocytosis, and extramedullary compartment) hematopoiesis, increased capacity od stem cell self- renewal, extramedulary hematopoiesis.

Tet2 KO Global loss of 5hmC and increase in 5mC in bone marrow [62] cells, chronic myelomonocytic leukemia - like phenotype at 2-4 months, splenomegaly and hepatomegaly, lethal myeloid malignancies

Maternal Tet3 KO Defects in paternal 5mC reprogramming, morphological [54] abnormalities at mid-gestation, reduced 5hmC levels, > 50% less viable pups at birth

Tet1 KO Loss of , smaller ovaries, small litter size, reduced [57, 59] fertility, impaired meiotic gene activation

Tet1/2 DKO Perinatal lethal (60%) with exencephaly, head [65] haemorrhage, growth retardation. 40% survivors display smaller ovaries, reduced fertility, decreased global 5hmC, partially compromised imprinting

Tet1 KO Reduced global 5hmC levels in the brain, impairment in [78] extinction, increased hippocampal long-term depression, down regulation of neuronal genes (Npas4, c- Fos, Egr2, Egr4, Npas4)

Tet1 KO Impaired spatial learning and memory, impaired [79] maintenance of the neural progenitor pool, defective adult

Tet1/3 KO Embryonic lethality, delayed early development, mitotic [63] defects, apoptosis, global loss of 5hmC and gain of 5mC, transcriptome variability, loss of Nanog expression, poor separation of germ layers

Tet1/2/3 TKO Complete embryonic lethality with severe gastrulation [60] defects, hyperactive Nodal signalling due to abnormal of Lefty1 and Lefty2 regulatory regions

Tet1/2/3 TKO Embryonic lethality, hyperactivation of Wnt singling in [41] neuromesodermal progenitors resulting in a skew towards mesoderm fate.

zebrafish tet1/2/3 TKO; Lethality following larval period, global reduction in 5hmC [66] (Danio tet1/2 DKO (> 30 fold) levels, smaller eyes, abnormal brain rerio) morphology, altered pigmentation at 36hpf, loss of differentiated definitive blood cells, aberrant Notch signalling in both tet1/2/3 TKO and tet1/2 DKO embryos

tet1/2/3 MO; tet3 Embryonic lethality (> 75%) with severe gastrulation [67] MO defects, short and blended axes, impaired head structures, small eyes and reduced pigmentation in tet1/2/3 MO. Minor defects such as microphthalmia in tet3 MO

Xenopus tet3 MO Eye malformations, microcephaly, reduced pigmentation, [11] laevis reduced expression of neural crest and neuronal markers,

Abbreviations 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; 5fC, 5-formylcytosine; 5caC, 5- carboxylcytosine; BER, ; DKO, double knock out; EB, embryoid body; ESC, embryonic stem cells; KD, knockdown; KO, knock out; LIF, Leukemia inhibitory factor; mESC, mouse embryonic stem cells; MO, morpholino; PRC2, polycomb repressive complex 2; TDG, thymine DNA glycosylase; TET, Ten-eleven translocation methylcytosine dioxygenase; TKO, triple knock out; siRNA, small interfering RNA; shRNA, small hairpin RNA; 2C - 2 cell.

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Figure 1. Structure and function of the Mus musculus TET protein family. All three TET family members display a conserved C-terminal domain that consists of a cysteine-rich region and a double-stranded β-helix domain (DSBH) domain, which harbours a low complexity region. The DBSH domain is responsible for iron ion binding and dioxygenease activity of TET proteins. Additionally, TET1 and TET3 exhibit a CXXC domain that is associated with binding to CpG dinucleotides. TET2 has lost its CXXC domain due to a chromosomal inversion, and the ancestral TET2 CXXC domain in mammals is encoded by a separate gene

(Idax). TET proteins can exist in isoforms with or without their associated CXXC domain, and such isoforms can be tissue- / cell type-specific [14, 80, 81].

Figure 1. Structure and function of the Mus musculus TET protein family. All three TET family members display a conserved C-terminal domain that consists of a cysteine-rich region and a double-stranded β-helix domain (DSBH) domain, which harbours a low complexity region. The DBSH domain is responsible for iron ion binding and dioxygenease activity of TET proteins. Additionally, TET1 and TET3 exhibit a CXXC domain that is associated with binding to CpG dinucleotides. TET2 has lost its CXXC domain due to a chromosomal inversion, and the ancestral TET2 CXXC domain in mammals is encoded by a separate gene (Idax). TET proteins can exist in isoforms with or without their associated CXXC domain, and such isoforms can be tissue- / cell type-specific [14, 80, 81].

Figure 2. Tet expression dynamics during embryoid body formation. Expression of Tet1, Tet2, and Tet3 in mESCs and embryoid bodies at four and eight days of differentiation. The dashed line tracks global 5hmC levels during this process.

Figure 3. A) TET protein function in vertebrates. Germ layer markers and transcription factors regulated by Tet1 and Tet3 during embryoid body differentiation [23, 41]. B) A unifying mechanism of TET protein function in vertebrates. TETs are responsible for demethylation and activation of regulatory elements associated with key signalling pathways during gastrulation and body plan formation in diverse vertebrates [41, 60, 67].