TET enzymes, DNA demethylation and pluripotency

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 genome. 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 reprogramming. 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.

Full text

TET enzymes, DNA demethylation and pluripotency
Samuel E Ross1, 2 and Ozren Bogdanovic1, 3
1 Genomics and Epigenetics 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: o.bogdanovic@garvan.org.au
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 genome. 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 reprogramming. 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 cytosines 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
transcription 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 gene expression 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 transcription factor
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 fertilisation 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
hydroxylation 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 germline-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.
!
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 embryonic development 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
shRNA KD
Impaired mESC self-renewal and maintenance, and
morphological abnormalities upon Tet1 KD
[5]
Tet1, Tet2, Tet3
siRNA KD; Tet1,
Tet2 shRNA KD
Global loss of 5hmC, increase in trophectoderm (Cdx2,
Eomes, Hand1), decrease in neuroectoderm (Pax6 and
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.
[23]
Tet1/2 siRNA KD;
Tet1 shRNA KD
Global loss of 5hmC, down-regulation of pluripotency
genes, increased expression of extraembryonic endoderm
differentiation markers, and increased Cdx2 expression
upon Tet1/Tet2 knockdown
[25]
Tet1 KO
Altered expression of lineage specification markers T and
Pax6, low efficiency of EB formation, loss of 5hmC and
trophectoderm-like cells in Tet KO teratomas
[37]
Tet1 siRNA KD
Loss of stem cell identity, morphological changes, global
5hmC loss, impaired LIF/Stat3 signalling, and upregulation
of differentiation markers
[38]
Tet1/2 DKO
Global reduction of 5hmC levels, trophectdoerm-like cells
in Tet1/2 DKO teratomas
[65]
Tet1/2/3 TKO
Global loss of 5hmC , defects in EB and teratoma
formation, poor contribution to chimeric embryos,
increased promoter 5mC, deregulation of developmental
genes
[35]
Tet1 TKO; Tet2 KO
hmC loss of 44% and 90,7% in Tet1 KO and Tet2 KO
respectively, increased 5mC on enhancers and delayed
gene induction during ES-NPC differentiation. in Tet2 KO,
[34]
Tet1/2/3 TKO
Lower (< 5%) global 5mC levels, increased 2C-like
population in Tet TKO ESCs
[36]

Tet3 KO; Tet1/2/3
TKO
Skewed differentiation toward cardiac mesoderm and
down-regulation of Wnt signalling inhibitor Sfrp4 during
differentiation of Tet3 KO mESCs, decreased
neuroectdoerm formation in Tet1/2/3 TKO
[41]
mouse
Tet1 KO
75% of homozygous pups displaying smaller body size
[37]
Conditional Tet2
KO (hematopoietic
compartment)
Enlargement of the hematopoietic stem cell compartment,
splenomegaly, monocytosis, and extramedullary
hematopoiesis, increased capacity od stem cell self-
renewal, extramedulary hematopoiesis.
[61]
Tet2 KO
Global loss of 5hmC and increase in 5mC in bone marrow
cells, chronic myelomonocytic leukemia - like phenotype at
2-4 months, splenomegaly and hepatomegaly, lethal
myeloid malignancies
[62]
Maternal Tet3 KO
Defects in paternal 5mC reprogramming, morphological
abnormalities at mid-gestation, reduced 5hmC levels, >
50% less viable pups at birth
[54]
Tet1 KO
Loss of oocytes, smaller ovaries, small litter size, reduced
fertility, impaired meiotic gene activation
[57, 59]
Tet1/2 DKO
Perinatal lethal (60%) with exencephaly, head
haemorrhage, growth retardation. 40% survivors display
smaller ovaries, reduced fertility, decreased global 5hmC,
partially compromised imprinting
[65]
Tet1 KO
Reduced global 5hmC levels in the brain, impairment in
memory extinction, increased hippocampal long-term
depression, down regulation of neuronal genes (Npas4, c-
Fos, Egr2, Egr4, Npas4)
[78]
Tet1 KO
Impaired spatial learning and memory, impaired
maintenance of the neural progenitor pool, defective adult
neurogenesis
[79]

Tet1/3 KO
Embryonic lethality, delayed early development, mitotic
defects, apoptosis, global loss of 5hmC and gain of 5mC,
transcriptome variability, loss of Nanog expression, poor
separation of germ layers
[63]
Tet1/2/3 TKO
Complete embryonic lethality with severe gastrulation
defects, hyperactive Nodal signalling due to abnormal
methylation of Lefty1 and Lefty2 regulatory regions
[60]
Tet1/2/3 TKO
Embryonic lethality, hyperactivation of Wnt singling in
neuromesodermal progenitors resulting in a skew towards
mesoderm fate.
[41]
zebrafish
(Danio
rerio)
tet1/2/3 TKO;
tet1/2 DKO
Lethality following larval period, global reduction in 5hmC
(> 30 fold) levels, smaller eyes, abnormal brain
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
[66]
tet1/2/3 MO; tet3
MO
Embryonic lethality (> 75%) with severe gastrulation
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
[67]
Xenopus
laevis
tet3 MO
Eye malformations, microcephaly, reduced pigmentation,
reduced expression of neural crest and neuronal markers,
[11]
Abbreviations
5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; 5fC, 5-formylcytosine; 5caC, 5-
carboxylcytosine; BER, base excision repair; 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].