PRIMER 1895
Development 139, 1895-1902 (2012) doi:10.1242/dev.070771
© 2012. Published by The Company of Biologists Ltd
Tet family proteins and 5-hydroxymethylcytosine in
development and disease
Li Tan1 and Yujiang Geno Shi1,2,*
Over the past few decades, DNA methylation at the 5-position
of cytosine (5-methylcytosine, 5mC) has emerged as an
important epigenetic modification that plays essential roles in
development, aging and disease. However, the mechanisms
controlling 5mC dynamics remain elusive. Recent studies have
shown that ten-eleven translocation (Tet) proteins can catalyze
5mC oxidation and generate 5mC derivatives, including 5hydroxymethylcytosine (5hmC). The exciting discovery of these
novel 5mC derivatives has begun to shed light on the dynamic
nature of 5mC, and emerging evidence has shown that Tet
family proteins and 5hmC are involved in normal development
as well as in many diseases. In this Primer we provide an
overview of the role of Tet family proteins and 5hmC in
development and cancer.
Key words: Tet, 5hmC, DNA methylation, Cancer
Introduction
DNA methylation at the 5-position of cytosine (5-methylcytosine;
5mC) is one of the key epigenetic marks that play a crucial role in
development and genome regulation (Bestor and Coxon, 1993;
Bird, 2002; Cedar and Bergman, 2009; Suzuki and Bird, 2008). It
has long been known that distinct genomic regions are differentially
methylated depending on cell or tissue type and developmental
stage (Suzuki and Bird, 2008). In addition to the establishment of
lineage-specific DNA ‘methylomes’ during mammalian
development, two waves of global DNA demethylation have also
been reported: one in the fertilized zygote and the other during
primordial germ cell development (Suzuki and Bird, 2008; Wu and
Zhang, 2010). The enzymes that catalyze DNA methylation,
namely the DNA methyltransferases (DNMTs) DNMT1,
DNMT3A, DNMT3B and the regulatory subunit DNMT3L, have
been identified and well characterized (Bestor et al., 1988;
Bourc’his et al., 2001; Okano et al., 1998). However, there remain
fundamental questions as to how genome-wide DNA methylation
is differentially regulated and dynamically processed during
development and to what extent the regulation of DNA methylation
might be linked to downstream gene expression programs that are
important for developmental processes.
The 5-hydroxymethylcytosine (5hmC) epigenetic mark was first
identified in the T-even bacteriophage almost six decades ago (Wyatt
and Cohen, 1953), and it was later found in the vertebrate brain and
in several other tissues (Globisch et al., 2010; Kriaucionis and Heintz,
2009; Nestor et al., 2011; Penn et al., 1972; Song, C. X. et al., 2011).
1
Laboratory of Epigenetics, Institutes of Biomedical Sciences, Fudan University,
Shanghai 200032, P. R. China. 2Division of Endocrinology, Diabetes, and
Hypertension, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
02115, USA.
*Author for correspondence ([email protected])
These studies suggested a significant biological role for 5hmC in
vertebrates. Interestingly, although 5hmC exists in mouse embryonic
stem (ES) cells at high levels, it decreases significantly after ES cell
differentiation (Szwagierczak et al., 2010; Tahiliani et al., 2009) but
rises again in terminally differentiated cells, such as Purkinje neurons
(Kriaucionis and Heintz, 2009). Very recently, it was shown that
5hmC exists in mouse, bovine and rabbit zygotes, and that 5hmC
accumulates specifically in the paternal pronucleus coinciding with
a reduction in 5mC (Iqbal et al., 2011; Wossidlo et al., 2011), which
suggests a potential biological function of 5hmC and a role in the
regulation of DNA methylation dynamics in early development.
Recently, human ten-eleven translocation 1 (TET1), a member
of the Tet family of proteins, was identified as a 5mC dioxygenase
responsible for catalyzing the conversion of 5mC to 5hmC
(Tahiliani et al., 2009). The mammalian Tet family contains three
members, Tet1, Tet2 and Tet3, all of which share a high degree of
homology within their C-terminal catalytic domain (Iyer et al.,
2009; Loenarz and Schofield, 2009). The discovery of this family
of enzymes suggested a potentially novel mechanism for the
regulation of DNA methylation, with 5hmC acting as an
intermediate during DNA demethylation, although the biology and
regulation of 5hmC and Tet family enzymes during development
remain elusive. Furthermore, it was shown that Tet proteins can
oxidize 5mC or 5hmC further and convert them to 5formylcytosine (5fC) and/or 5-carboxylcytosine (5caC) (He et al.,
2011; Ito et al., 2011), adding another layer of complexity to
uncovering the potential function of Tet family enzymes and 5hmC
in epigenetic regulation during development. In this Primer, we
provide an overview of Tet family proteins, focusing on the
developmental roles of Tet proteins and 5hmC in mice. We also
summarize the emerging roles for Tet proteins in human cancers.
Domain structure of Tet family proteins
The mouse Tet gene family has three members: Tet1, Tet2 and Tet3
(Ito et al., 2010; Tahiliani et al., 2009). All Tet proteins contain a
catalytic C-terminal CD domain (Cys-rich and DSBH regions) that
belongs to the Cupin-like dioxygenase superfamily and exhibits 2oxoglutarate (2-OG)- and iron (II)-dependent dioxygenase activity
(Fig. 1). Tet proteins oxidize 5mC into 5hmC via these CD
domains and require a-ketoglutarate as a co-substrate for
enzymatic activity (Tahiliani et al., 2009). Recently, using
radiolabeled thin-layer chromatography and high-sensitivity
HPLC/mass spectrometry assays, Zhang and colleagues
demonstrated that the CD domain of Tet proteins can also oxidize
5mC and 5hmC into 5fC and/or 5caC, although at very low levels
(Ito et al., 2011). In addition, Xu and colleagues reported that Tet
proteins can substantially oxidize 5mC and 5hmC into 5caC via
their CD domains in the presence of ATP in vitro (He et al., 2011).
Another distinct feature of Tet family proteins is the CXXC zincfinger domain, which was first identified and defined in DNMT1
(Bestor, 1992). A CXXC domain can be found in the N-terminus
DEVELOPMENT
Summary
1896 PRIMER
Development 139 (11)
CD
Tet2
1912 a.a.
CD
Tet3
1668 a.a.
CD
Key
CXXC (binds CpG islands)
Cys-rich (methylcytosine dioxygenase activity)
DSBH (methylcytosine dioxygenase activity, metal binding)
Spacer region (unknown function)
PRK12323 (unknown function)
Fig. 1. Structure and function of mouse Tet family proteins. The
mouse Tet protein family contains three members: Tet1, Tet2 and Tet3.
All have a C-terminal CD domain (containing the Cys-rich and DSBH
regions) that exhibits 2-oxoglutarate (2-OG)- and iron (II)-dependent
dioxygenase activity. The CD domain also includes a spacer region, the
length of which varies between Tet family members. The N-termini of
Tet1 and Tet3, but not Tet2, contain a CXXC domain, which mediates
their direct DNA-binding ability (Zhang et al., 2010; Xu, Y. et al., 2011).
There is also a unique PRK12323 (DNA polymerase III subunits gamma
and tau; provisional) domain in Tet3. This PRK12323 domain is not
assigned to any domain superfamily and its function remains unknown.
a.a., amino acids.
of Tet1 and Tet3 but not in Tet2 (Fig. 1). Unlike the CXXC
domains in other proteins [for example, DNMT1,
myeloid/lymphoid or mixed-lineage leukemia (MLL) and CXXC
finger protein 1 (CFP1, or CXXC1)], which are known to bind
unmethylated CpG dinucleotides (Cierpicki et al., 2010; Song, J. et
al., 2011; Xu, C. et al., 2011), the function of this domain in Tet1
and Tet3 is largely unknown. It is known, however, that the CXXC
domain of TET1 recognizes not only unmodified cytosine but also
5mC and 5hmC, and it prefers to bind to regions in the genome of
high CpG content (Zhang et al., 2010; Xu, Y. et al., 2011).
Consistent with this feature, genome-wide mapping of Tet1 binding
by ChIP-seq approaches revealed its enrichment around
transcription start sites (TSSs) in mouse ES cells (Xu, Y. et al.,
2011; Williams et al., 2011; Wu et al., 2011b). Although future
investigation is required to elucidate the importance of this domain
in Tet1 or Tet3 function, we are tempted to speculate that the
CXXC domain serves as the functional unit that targets these
enzymes to specific genomic regions for their action.
In addition to the above-described functional domains
responsible for executing Tet protein action, there is a spacer region
that bridges the two parts of the disconnected DSBH enzymatic
domain. This unique spacer region is common to all Tet family
members, although its length varies (Fig. 1). The functional
importance of this spacer region is currently unknown.
Interestingly, Upadhyay et al. revealed that the spacer region of
Tet1 has significant sequence similarity to the C-terminal domain
(CTD) of RNA polymerase II of Saccharomyces cerevisiae
(Upadhyay et al., 2011). Although not identical, many key residues
important for post-translational protein modification, such as
phosphorylation and methylation, are invariant and conserved
between the two sequences. Furthermore, TET2 is one of the most
frequently mutated genes in myelodysplastic syndrome (MDS)
(Kosmider et al., 2009), and about one-quarter of mutations were
found in the region corresponding to the spacer domain of TET2,
highlighting the functional importance of this region (Ko et al.,
2010). Although future structural, deletion/mutational and/or
biochemical analyses are required to uncover its function, it is
possible that the spacer region of Tet family proteins, similar to the
CTD of RNA polymerase II, is post-translationally modified, which
might in turn play a ‘switch’ role in orchestrating the complex
action of Tet proteins on 5mC or during targeted gene regulation.
In summary, these studies have shown that Tet family proteins,
via their CD domains, possess an intrinsic enzymatic capability to
convert 5mC to 5hmC, 5fC or 5caC depending on the presence of
distinct co-factors such as ATP (Fig. 2). Tet proteins also contain a
number of additional domains with as yet undefined functions.
However, it appears that the Tet family proteins alone are not able
to complete the demethylation of 5mC to unmodified cytosine.
Mechanism of action of Tet proteins: a role for
5hmC in the regulation of DNA methylation and
gene expression
Recent studies suggest that there might be multiple pathways or
mechanisms by which 5hmC and Tet family proteins regulate
DNA methylation dynamics and gene transcription (Fig. 3). Song
and colleagues presented a model in which 5hmC is more sensitive
than 5mC to deamination by activation-induced deaminase (AID),
and the deamination product, 5-hydroxymethyluracil (5hmU),
activates base-excision repair (BER) pathway-mediated
demethylation (Guo et al., 2011), suggesting one route to achieve
Tet protein-mediated active DNA demethylation (Fig. 3).
Furthermore, recent findings have shown that Tet proteins can
oxidize 5mC not only to 5hmC but also 5fC and 5caC, which can
subsequently be recognized and excised by thymine DNA
glycosylase (TDG) in vitro and in vivo (He et al., 2011; Ito et al.,
2011; Pfaffeneder et al., 2011; Zhang et al., 2012). In addition to
these mechanisms of active DNA demethylation, the recent finding
that 5hmC, 5fC and 5caC associated with the paternal genome in
the zygote are gradually lost during preimplantation development
suggested a dominant mechanism of passive DNA demethylation
at this developmental stage (Inoue and Zhang, 2011; Inoue et al.,
2011). Although these studies propose alternative routes for Tetmediated active or passive DNA demethylation, it is suggested that
the initial oxidation of 5mC to 5hmC by Tet family proteins is a
prerequisite for the subsequent demethylation processes, regardless
of how these final steps are mediated (e.g. by deamination, BER
or TDG action), to complete the cycle of DNA demethylation (He
et al., 2011; Ito et al., 2011; Maiti and Drohat, 2011). These studies
thus suggest that 5hmC acts as an intermediate during DNA
demethylation and that Tet proteins might function as DNA ratelimiting demethylation regulators. However, these studies also
raise a number of questions (see Box 1). For example, why has
5hmC been detected as a relatively stable epigenetic mark in ES
cells as well as in many normal tissues if the primary role of 5hmC
is as an intermediate of 5mC demethylation? Why are 5fC and
5caC much less abundant than 5hmC in the investigated cells and
tissues if they are also intermediates of 5mC demethylation? It is
of interest to investigate how the extent of Tet-catalyzed 5mC
oxidation is regulated in vivo and whether there exist DNA
decarboxylases for 5caC or DNA deformylases for 5fC.
Alternatively, 5hmC might also function as a new landmark in the
epigenetic landscape and may recruit specific readers that
subsequently direct the dynamic remodeling and organization of the
chromatin structures that are important for proper gene transcription.
Although many methyl-CpG-binding domain (MBD) proteins [such
as MBD1, MBD2, methyl CpG-binding protein 2 (MECP2) and
DEVELOPMENT
Tet1
2036 a.a.
PRIMER 1897
Development 139 (11)
TET1/2/3
Fe(II)
2-OG, O2
5mC
TET1/2/3
Fe(II), ATP
Succinate, CO2
2-OG, O2
5hmC
TET1/2/3
Fe(II), ATP
Succinate, CO2
2-OG, O2
5fC
Succinate, CO2
5caC
Fig. 2. Oxidative reactions carried out by Tet proteins. Tet family proteins oxidize 5mC into 5hmC (and to 5fC and 5caC at low levels) in the
presence of 2-OG and iron. 5mC and 5hmC can be further oxidized mainly to 5caC by Tet proteins if the reaction system is supplemented with
ATP. 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; 5fC, 5-formylcytosine; 5caC, 5-carboxylcytosine.
performed by other laboratories did not affect Nanog expression or
the self-renewal of mouse ES cells (Dawlaty et al., 2011; Koh et
al., 2011; Williams et al., 2011; Xu, Y. et al., 2011). Thus, the
involvement of Tet family proteins and 5hmC in ES cell selfrenewal or pluripotency can be determined only if we can clearly
illustrate the mechanism by which 5hmC and Tet are involved in
the regulation of those critical genes.
Box 1. Tet proteins and 5hmC in development and
cancer: open questions
Tet-mediated DNA demethylation: active or passive?
Active DNA demethylation occurs during at least two stages of
mouse development: in fertilized zygotes and in primordial germ
cells (PGCs). In fertilized zygotes, Tet3-mediated 5mC oxidation can
lead to active demethylation (with the help of some DNA repair
enzymes) or may result in passive demethylation due to rapid DNA
replication in the early embryo. Do these mechanisms also apply to
the global demethylation that occurs in PGCs? Are there
decarboxylases that directly convert 5caC to unmodified cytosine
to complete the demethylation of 5mC?
What are the 5hmC readers?
Many 5mC-binding proteins and other proteins function as readers
of 5mC, whereas little is known about how the 5hmC mark is
recognized. Does a true 5hmC-specific reader exist? The
identification of 5hmC-specific binding proteins will help to
decipher the functions of 5hmC and 5mC.
How do the catalytic activity-independent functions of Tet
proteins contribute to their role?
Recent work has revealed that Tet1 can exert catalytic activityindependent functions. How Tet proteins act to regulate gene
transcription in a catalytic activity-independent fashion needs to be
investigated, as does the distinction between the catalytic activitydependent and -independent functions of Tet proteins during
mouse development and in disease.
Are there evolutionary roles for 5hmC?
As an epigenetic modification based on 5mC, 5hmC might have a
less fundamental role than its precursor and might rather serve to
increase the complexity of DNA methylation regulation in
mammals. Does 5hmC share the same history as 5mC during
evolution? When did Tet proteins and 5hmC appear in evolutionary
history? Given that brain tissues exhibit the highest levels of 5hmC
in adult mice, have 5hmC and Tet proteins contributed to the
increasingly complex function (such as in neural circuitry and
behaviors) of mammals during evolution?
How does 5hmC function in cancer?
Evidence suggests that 5hmC is greatly reduced or suppressed in
multiple cancers. However, what is the role of 5hmC in cancer
prevention and/or tumorigenesis? How is 5hmC downregulated in
cancers? Mechanistically, answering these questions might offer new
strategies for anti-cancer drug development and cancer therapy.
DEVELOPMENT
MBD4] do not bind 5hmC in vitro (Jin et al., 2010), a recent study
proposed that Tet1 is associated with the Mbd3/NuRD complex in
mouse ES cells and that Mbd3 can bind 5hmC and unmodified
cytosine, but not 5mC, in vitro (Yildirim et al., 2011). Given that
most, if not all, 5hmC comes from 5mC (Ficz et al., 2011),
establishing DNA methylation at the hydroxymethylated sites during
DNA replication appears to be a prerequisite for 5hmC maintenance.
UHRF1 (ubiquitin-like, containing PHD and RING finger domains
1) is required for maintenance of methylation by interacting with
DNMT1 and hemi-methylated CpG (Bostic et al., 2007; Sharif et al.,
2007). The finding that UHRF1 can bind both 5mC and 5hmC
(Frauer et al., 2011) provides a possible mechanism by which
UHRF1 may also target DNMT1 to the hemi-hydroxymethylated
replication forks. More recently, Cheng and colleagues showed that
DNMT1 has a high intrinsic activity toward the hemi-methylated
CpG substrate but a greatly reduced activity for the hemihydroxymethylated CpG substrate in methyl transfer assays in vitro
(Hashimoto et al., 2012). Interestingly, they found that DNMT3A
and DNMT3B exhibited equal activities on unmodified, hemimethylated and hemi-hydroxymethylated CpG substrates
(Hashimoto et al., 2012). Collectively, these studies suggest that,
instead of being merely an intermediate of DNA demethylation,
5hmC acts as a stable epigenetic mark that directly influences
genome structure and function. Future experiments that aim to
identify additional specific 5hmC ‘readers’ and the cellular
complexes associated with 5hmC should distinguish between 5mC
and 5hmC in recruiting their specific readers (see Box 1). In addition,
such studies will provide new insight into our understanding of the
multiple mechanisms of action of 5hmC in the dynamics of DNA
methylation and chromatin structure.
Although 5hmC has been implicated as either a stable epigenetic
modification (the ‘sixth base’) or an intermediate of DNA
demethylation, correlation between genome-wide 5hmC
enrichment and gene transcription activity in mouse ES cells and
brain tissue remains elusive and the findings remain somewhat
controversial (Ficz et al., 2011; Jin et al., 2011a; Pastor et al., 2011;
Robertson et al., 2011; Song, C. X. et al., 2011; Stroud et al., 2011;
Williams et al., 2011; Wu et al., 2011a; Xu, Y. et al., 2011). Zhang
and colleagues showed that the knockdown of Tet1 by shRNA can
increase the 5mC level on Nanog promoters and repress the
transcription of Nanog mRNA, which subsequently skews the
mouse ES cells toward a differentiated state (Ito et al., 2010). A
recent study also showed that acute depletion of Tet1-dependent
5hmC levels by shRNA impairs leukemia inhibitory factor
(LIF)/Stat3 signaling and results in a loss of ES cell identity. This
supports the idea that Tet1 is essential for the regulation of key
genes involved in ES cell self-renewal and lineage determination
(Freudenberg et al., 2011). However, knockout of Tet1 by
homologous recombination or knockdown of Tet1 by shRNA
1898 PRIMER
Development 139 (11)
Tet member serve as potential molecular mechanisms that
differentially contribute to the regulation of DNA methylation
status and gene expression. Future studies are needed to clarify
how Tet proteins are regulated by their post-translational
modifications or their associated co-factors. Furthermore, the
spacer region of Tet family proteins is currently understudied. Its
potential regulatory function, e.g. its role in orchestrating the
formation of Tet complexes and modulating Tet enzymatic
activities, warrants future investigation.
Decarboxylase?
5caC
TDG
BER
TET1/2/3
Deformylase?
5fC
TET1/2/3
BER
5hmU
AID
5hmC
TET1/2/3
Cytosine
DNMT1, DNMT3A/B
5mC
AID
BER
Thymine
Passive demethylation during DNA replication
Fig. 3. Involvement of Tet family proteins in the regulation of
DNA methylation and demethylation: potential mechanisms. Tet
family proteins (TET1/2/3) catalyze 5mC oxidation to 5hmC. Tet family
proteins can also convert 5mC or 5hmC further into 5fC and 5caC, and
the latter may be recognized and excised by thymine DNA glycosylase
(TDG) to generate cytosine and thereby complete demethylation.
Alternatively, because 5hmC is more sensitive than 5mC to
deamination (via activation-induced deaminase, AID), it can be
converted to 5-hydroxymethyluracil (5hmU), which in turn can be
converted to cytosine following base-excision repair (BER) pathwaymediated demethylation (Guo et al., 2011). It remains unknown
whether there are decarboxylases or deformylases that can remove the
modification directly. Importantly, 5mC and its oxidative derivatives
may undergo passive demethylation by dilution during DNA replication
(Inoue and Zhang, 2011; Inoue et al., 2011).
Finally, although many studies have indicated that the enzymatic
activity of the Tet family proteins is important for their function, a
recent study suggested that Tet proteins might also exert functions
independently of their catalytic activity (Williams et al., 2011). In
that study, Helin and colleagues demonstrated that Tet1 associates
and colocalizes with the Sin3a co-repressor complex in 293T and
mouse ES cells (Williams et al., 2011). Importantly, they found that
the upregulation of Tet1 target genes upon Tet1 knockdown can
also be observed in DNMT triple knockout ES cells in which both
5mC and 5hmC modifications are absent (Williams et al., 2011).
These results suggest that Tet1 might repress gene transcription in
a catalytic activity-independent manner.
Taken together, it is likely that different Tet family members are
involved in the regulation of DNA methylation and/or gene
expression via different mechanisms. The multi-protein complexes
with which the Tet family members associate, the DNA-binding
activity of the CXXC domain, or the dioxygenase activities of each
The role of Tet family proteins and 5hmC during
mouse development
Dramatic epigenomic reprogramming, including DNA methylation
and demethylation, is essential for mammalian development.
Recently, the developmental roles of Tet family proteins and 5hmC
have been investigated using gene knockout strategies (see Table
1). Below, we summarize the function of Tet family proteins at
different stages throughout development.
Tet proteins in primordial germ cells and gametes
Although embryonic development starts with oocyte fertilization, the
oocyte and sperm develop from primordial germ cells (PGCs).
Mouse PGCs are specified from epiblast cells and migrate into the
gonad region at embryonic day (E) 7.25. This is followed by
widespread epigenetic reprogramming at E11.5, which includes
genome-wide DNA demethylation, erasure of genomic imprints, and
large-scale chromatin remodeling (Hajkova et al., 2008; Kota and
Feil, 2011). Both Tet1 and Tet2 are expressed in E11.5 and E12.5
PGCs but less so in oocytes and zygotes, whereas Tet3 is highly
expressed in oocytes but not in PGCs (Gu et al., 2011; Hajkova et
al., 2010; Surani and Hajkova, 2010). The development of PGCs,
and subsequently of spermatocytes or oocytes, is normal in Tet1 or
Tet2 knockout mice, and these knockout adults display similar
fecundity to wild-type mice (Dawlaty et al., 2011; Li et al., 2011; Ko
et al., 2011; Moran-Crusio et al., 2011; Quivoron et al., 2011). Xu
and colleagues established PGC-conditional Tet3 knockout mice and
found that oogenesis and spermatogenesis are normal when Tet3 is
absent in PGCs (Gu et al., 2011). These findings support the
conclusion that, despite the dramatic reprogramming events that
occur in PGCs and gametes, the knockout of Tet1, Tet2 or Tet3 alone
has no significant effect on the development of PGCs and gametes.
Tet proteins in the zygote
DNA demethylation also occurs in the zygote, when the paternal
genome loses its DNA methylation marks rapidly after fertilization
(Oswald et al., 2000). It was shown recently that 5mC in the
paternal but not the maternal pronucleus is oxidized to 5hmC (Gu
et al., 2011; Iqbal et al., 2011; Wossidlo et al., 2011). The
expression levels of Tet1 and Tet2 are very low in oocytes or
Table 1. Phenotypes of Tet knockout mice
Preimplantation
development
Postimplantation
development
Postnatal development
Tet1
Tet2–/–
Normal
Normal
Small body size
Normal
Small body size
Spontaneous myeloid
leukemia
Tet3–/–
Tet3mat–/pat+
Normal
Normal, but blockage in
paternal genome
reprogramming
Normal
A high frequency of
degeneration and
variable multi-organ
abnormalities
Neonatal lethality
Neonatal lethality and small
body size
–/–
References
(Dawlaty et al., 2011)
(Ko et al., 2011; Li et al., 2011;
Moran-Crusio et al., 2011; Quivoron
et al., 2011)
(Gu et al., 2011)
(Gu et al., 2011)
DEVELOPMENT
Genotype
PRIMER 1899
Development 139 (11)
Tet2
Tet3
5mC
5hmC
Zygote
Two-cell
Four-cell
Blastocyst
Fig. 4. Tet gene expression and 5mC and 5hmC levels during
mouse preimplantation development. The expression levels of Tet1
and Tet2 are very low in oocytes and fertilized zygotes. By contrast,
Tet3 is highly expressed in oocytes and fertilized zygotes but disappears
rapidly during cleavage (Gu et al., 2011; Iqbal et al., 2011; Wossidlo et
al., 2011). Tet3-mediated 5mC oxidation and subsequent dilution by
DNA replication during embryo cleavage might contribute to the
previous ‘active’ DNA demethylation observed in the developing
preimplantation embryo (Gu et al., 2011; Inoue and Zhang, 2011). The
expression levels of Tet1 and Tet2 increase during preimplantation
development and both are highly expressed in the inner cell mass of
the blastocyst, which also exhibits high 5hmC levels (Ruzov et al.,
2011).
fertilized zygotes (Gu et al., 2011; Iqbal et al., 2011; Wossidlo et
al., 2011). By contrast, Tet3 is highly expressed in oocytes and
fertilized zygotes but disappears rapidly during cleavage (Gu et al.,
2011; Iqbal et al., 2011; Wossidlo et al., 2011). In line with this, Xu
and colleagues showed recently that maternal Tet3 is the key
enzyme catalyzing 5mC into 5hmC in the paternal genome
(paternal pronuclei) of developing zygotes (Gu et al., 2011). DNA
demethylation and 5hmC generation can also occur in the maternal
pronuclei of Stella (Dppa3) null zygotes, suggesting that the Stella
protein protects the maternal pronucleus from DNA demethylation
and oxidation (Nakamura et al., 2007; Wossidlo et al., 2011).
Similarly, Tet3-mediated 5mC oxidation in oocytes is also required
for epigenetic reprogramming of the donor nuclear DNA during
somatic cell nuclear transfer (Gu et al., 2011). Furthermore, Zhang
and colleagues observed that 5hmC and other 5mC derivatives can
be simply eliminated by passive dilution through DNA replication
during the division of zygotes (Inoue and Zhang, 2011; Inoue et al.,
2011). It is conceivable that converting 5mC to 5hmC via Tet3mediated oxidation is a key step in initiating the zygotic ‘resetting’
of DNA methylation patterns, while subsequent passive dilution of
5hmC through DNA replication during zygote cleavage serves as
one of the mechanisms for ultimate erasure of paternal 5mC (Fig.
4). Nonetheless, although these new findings shed light on the
molecular mechanisms that underlie the first wave of DNA
demethylation in the earliest developmental stage of life, it remains
unclear to what extent 5mC oxidation, followed by passive DNA
demethylation, accounts for the demethylation of paternal
pronuclear DNA in the zygote.
Tet proteins in pre- and postimplantation embryos and in
ES cells
Both Tet1 and 5hmC levels are abundant in mouse ES cells, in the
inner cell mass of the mouse blastocyst, and in cells of the early
mouse epiblast (Ito et al., 2010; Koh et al., 2011; Ruzov et al.,
2011). Recent work from Ito et al. found that the shRNA-mediated
knockdown of Tet1, but not Tet2 or Tet3, led to the spontaneous
differentiation of mouse ES cells even in the presence of LIF (Ito
et al., 2010). However, others believe that Tet1 is not required for
maintaining the self-renewal of mouse ES cells (Koh et al., 2011;
Williams et al., 2011; Xu, Y. et al., 2011); recently, Jaenisch and
colleagues found that Tet1 null ES cells maintain their self-renewal
ability under mouse ES cell culture conditions (LIF plus fetal
bovine serum) in vitro and develop normally in vivo (Dawlaty et
al., 2011). Furthermore, whereas both knockdown and knockout of
Tet1 force the transdifferentiation of mouse ES cells into
trophoblasts in vitro, such effects were not detected in vivo
(Dawlaty et al., 2011; Ito et al., 2010; Koh et al., 2011), and Tet1–/–
and Tet2–/– mice appear to develop normally, appear healthy
through adulthood and are fertile (Dawlaty et al., 2011; Ko et al.,
2011; Li et al., 2011; Moran-Crusio et al., 2011; Quivoron et al.,
2011) (Table 1). It is still unclear why Tet1 expression and 5hmC
are maintained at high levels in mouse ES cells. As such, the
biological role of Tet1/Tet2 or 5hmC in postimplantation
embryonic development remains unclear and controversial.
One explanation for the discrepancies between different research
groups regarding the functional roles of Tet1 and 5hmC in
postimplantation embryonic development might be that other Tet
proteins play compensatory roles, as knockout or knockdown of
Tet1 or Tet2 results in only a partial decrease in 5hmC levels
(Dawlaty et al., 2011; Ito et al., 2010; Koh et al., 2011; Xu, Y. et
al., 2011). Interestingly, homozygous mutation of Tet3 led to
neonatal lethality, highlighting its importance in the development
of multiple organs during embryogenesis (Gu et al., 2011) (Table
1). Thus, Tet family proteins may have important yet redundant
roles in the precise regulation of postimplantation embryonic
development.
Tet proteins in postnatal development
Although the genomic DNA sequence is the same, each organ or
tissue in the mouse has a tissue-specific DNA methylome (Liang
et al., 2011; Previti et al., 2009). As a derivative of 5mC, 5hmC can
be detected in almost all organs and tissues, but unlike the
relatively constant levels of 5mC, 5hmC levels vary among tissues
(Ito et al., 2010; Globisch et al., 2010; Li et al., 2011). For example,
in the brain, genome-wide analysis of 5hmC distribution and
localization revealed that 5hmC levels in the cerebellum and
hippocampus of adult mice (6 weeks and 1 year of age) are higher
than those observed in postnatal day (P) 7 mice (Szulwach et al.,
2011). MECP2 is a methyl-CpG-binding protein that plays an
important role in neural development and brain function (Lewis et
al., 1992). Accordingly, mutations in human MECP2 cause the
neurodevelopmental disorder Rett syndrome (Amir et al., 1999).
Using Mecp2 knockout and knockin mice, Szulwach et al. found
that the overall abundance of 5hmC in the cerebellum was
negatively correlated with Mecp2 dosage (Szulwach et al., 2011).
These studies suggested that 5hmC dynamics might be involved in
postnatal neurodevelopment and brain function and, when
abnormal, may lead to neural disorders. Tet family genes are
expressed in multiple regions of the brain and therefore it will be
worthwhile to carefully analyze the brain structures,
neurophysiology and behaviors of Tet knockout mice to uncover
the potential function of Tet proteins in brain development,
function and pathology (see Box 1).
Recent evidence suggests that Tet2 and 5hmC are also associated
with postnatal development of the hematopoietic lineage, and
alteration of Tet2 expression or the levels of 5hmC may disrupt the
homeostasis of cells and ultimately lead to abnormalities in
DEVELOPMENT
Tet1
hematopoietic cell proliferation and maturation (Abdel-Wahab et
al., 2009; Albano et al., 2011; Chou et al., 2011; Euba et al., 2011;
Figueroa et al., 2010; Pronier et al., 2011; Weissmann et al., 2011).
Although Tet2–/– mice appeared to develop normally overall, they
displayed an abnormality in myeloid lineage differentiation and
developed leukemia spontaneously (Ko et al., 2011; Li et al., 2011;
Moran-Crusio et al., 2011; Quivoron et al., 2011). Since the Tet3–/–
phenotype was neonatal lethal, the exact functions of Tet3 protein
in postnatal development are largely unknown and need further
investigation (Gu et al., 2011). We believe that conditional
knockout of Tet3 in various tissues or organs postnatally will
provide a good strategy for probing the biological role of Tet3
during mouse postnatal development.
Tet family genes and 5hmC in human cancers
TET1 was originally discovered as a gene that, as result of a
chromosome translocation, was fused to the MLL gene in certain
leukemia patients (Lorsbach et al., 2003). However, the role of
TET1 in hematopoietic development and leukemogenesis remains
ambiguous. By contrast, TET2, although only recently identified,
is the most studied member of the Tet family genes in MDS and
other types of leukemia. It has been reported as one of the most
frequently mutated genes in myeloid malignancies (Abdel-Wahab
et al., 2009; Albano et al., 2011; Chou et al., 2011; Euba et al.,
2011; Figueroa et al., 2010; Weissmann et al., 2011). Whereas
some of the mutation sites in TET2 correspond to residues within
the catalytic domain and could thus impair catalytic activity, many
mutations appear unrelated to enzymatic activity (Ko et al., 2010).
Therefore, the molecular mechanism underlying TET2 loss-offunction in leukemogenesis is still unknown and requires further
investigation.
So far, a direct correlation between TET3 and cancer has not
been reported. A possible explanation for this is that, among the
three Tet proteins, TET3 is the most important regulator and critical
TET3 mutations or dysregulation might lead to lethality. Besides
the hematopoietic malignancies, 5hmC levels are decreased in a
broad range of solid tumors, including glioma, colon cancer, breast
cancer and melanoma (Li and Liu, 2011; Jin et al., 2011b; Haffner
et al., 2011; Xu, W. et al., 2011). Since global DNA
hypomethylation and locus-specific DNA hypermethylation have
been identified as key features of many cancers (Feinberg et al.,
2006; Suzuki and Bird, 2008), investigating whether low 5hmC
levels contribute to aberrant DNA methylation patterns or other
aspects in cancers could prove to be very informative (see Box 1).
Conclusions
The role of Tet proteins and 5hmC in development and disease has
been widely studied in the past three years since the first
biochemical connection between Tet proteins and 5hmC was
established. In the future, studies using conditional knockouts of
Tet genes will decipher the stage- and organ-specific functions of
Tet proteins and 5hmC in development. Double or triple knockout
of Tet genes may circumvent their redundant/compensatory effects
on 5mC oxidation and gene regulation, offering a better
understanding of the role of 5hmC and Tet family genes in
development. Moreover, further investigations are needed to
distinguish the catalytic activity-dependent and -independent
functions of Tet proteins. Since most studies of Tet proteins and
5hmC have focused on mice, rats and humans, expanding these
investigations to other model systems (such as Xenopus, zebrafish
and even non-vertebrates) will elucidate the evolutionally
conserved functions of these proteins. Finally, although the current
Development 139 (11)
data from solid and hematopoietic tumors support a role for 5hmC
as an epigenetic modification requisite for preventing
tumorigenesis, it remains unknown whether the decrease in 5hmC
level is a cause or a consequence of preventing tumorigenesis. In
summary, many important issues need to be explored in the future
(see Box 1). Clearly, the roles and mechanisms of action of 5hmC
and Tet family members in ES cell biology and during embryonic
development, as well as in cancer biology, will be the main areas
of focus. In the next five years, new and exciting discoveries in
these areas are expected to emerge.
Acknowledgements
We thank members of the Y.G.S. laboratory for helpful discussion and
Abraham Khorasani for critical reading of the manuscript. We apologize to
those colleagues whose publications could not be cited owing to space
limitations.
Funding
Work in the Y.G.S. laboratory is supported by the National Institutes of Health
and partly by the ‘985⬘ Project of the Ministry of Education of China and ‘973⬘
National Basic Research Program of China. Y.G.S. is a Pew scholar. Deposited
in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
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