TETs/hmC in cell reprogramming and differentiation
The TET proteins participate in two waves of global DNA demethylation that occur during reprogramming of primordial germ cells as well as of paternal pronucleus in the zygote. Such reprogrammings are needed to ensure resetting of the epigenomes for pluripotency (Gu et al, 2011; Inoue et al, 2011; Iqbal et al, 2011; Wossidlo et al, 2011; Hackett et al, 2012; Nakamura et al, 2012). When PGCs migrate to the future gonads, high levels of TET1 and TET2 allows conversion of 5mC into 5hmC and this, not only globally but also more locally (e.g. at genomic imprinted loci). This is followed by subsequent passive “replication-coupled” dilution of 5hmC during several round of cell divisions with a progressive loss of hydroxymethylation (Hackett et al, 2012). A second wave of global DNA demethylation appears when the spermatozoon fertilizes the oocyte. In this case, TET3 hydroxylates the paternal DNA while PGC7 (also called DPPA3/STELLA) maintains normal methylation levels on the maternal genome as well as on imprinted genes by binding to the repressive H3K9me2 histone mark and by repelling TET3 (Szabo & Pfeifer, 2012). Worth mentioning, it has been observed that in the paternal pronucleus, 5hmC is further oxidized into 5fC and 5caC and that those modifications are also passively diluted through cell divisions (Gu et al, 2011; Inoue & Zhang, 2011; Inoue et al, 2011; Iqbal et al, 2011; Wossidlo et al, 2011; Nakamura et al, 2012).
The initial discovery of TET enzymes in ESCs has led many groups to assess their importance in pluripotency maintenance and cell differentiation. Although some reports suggest a role of TET1 in maintenance of pluripotency, especially via direct binding and demethylation of the NANOG promoter, other papers do not support this view (Ito et al, 2010; Ficz et al, 2011; Koh et al, 2011; Williams et al, 2011a; Wu et al, 2011; Xu et al, 2011b; Doege et al, 2012; Freudenberg et al, 2012). Furthermore, in vivo results obtained with TET1 knockout and TET1/TET2 double-knockout mice (under conditions where TET3 is unlikely to compensate significantly for the knockout) suggest that ESCs stemness can be maintained without these methyldioxygenases (Dawlaty et al, 2011, 2013). In fact, although a fraction of the double KO mice displayed midgestation abnormalities and perinatal lethality, viable and fertile mice were also obtained. Those however display a reduced size and weight as compared to their wt counterparts and the females also show smaller ovaries and reduced fertility. This indicates an overtly normal development with variable phenotypes probably due to variable penetrance of epigenetic defects. This double knockout is in sharp contrast with the TET3 knockout embryos from Xu's group that show apparent lethality either around embryonic day 11.5 or after birth, highlighting the crucial role of TET3 in reprogramming the paternal pronucleus after fertilization (Gu et al, 2011). While the jury is still out on whether TETs play a role in maintaining pluripotency in ESCs, another phenomenon involving TETs is coming to light: reprogramming of developmentally committed cells such as MEFs to produce induced pluripotent stem cells (iPSCs). iPSCs, phenotypically comparable to ESCs, can be produced in vitro upon overexpression of the Yamanaka “OSKM” factors (OCT4, SOX2, KLF4 and C-MYC). During this process profound epigenetic reprogramming takes place, with loss of methylation on the promoters of several pluripotency genes (Takahashi & Yamanaka, 2006). Several studies have stressed the importance of TETs in iPSC reprogramming. In the first of these studies, TET2 and PARP1 were identified as key factors in this process: both are upregulated in MEFs upon OSKM treatment, and both bind to ESRRB and NANOG to shape the chromatin landscape essential for correct gene expression and iPSC reprogramming (Doege et al, 2012). As mentioned earlier, another report revealed that NANOG can interact with TET1 and TET2 and facilitate reprogramming of neural stem cells in a TET-activity-dependent manner (Costa et al, 2013). Remarkably, Gao et al (2013) were able to induce iPSC formation in experiments where they replaced OSKM with “TSKM” (TET1, SOX2, KLF4, and C-MYC), probably because OCT4 is a direct binding target of TET1. These results highlight the importance of TET1 and TET2 in the demethylation and activation of key pluripotency genes such as ESRRB, NANOG, and OCT4 during reprogramming of differentiated cells, and thus suggest an important role for methyldioxygenases in regenerative medicine studies.
Recent reports pinpoint a participation of TETs in terminal differentiation as well. In 2013, Hahn et al found the 5hmC pattern to be very dynamic during NPC differentiation to neurons. In gene bodies they observed a global increase in 5hmC without any subsequent change in DNA methylation. They further showed that TET2 and TET3 are required for correct NPC differentiation and that their loss leads to abnormal accumulation of cell clusters along the radial axis in the intermediate and ventricular zones (Hahn et al, 2013). A similar increase in hydroxymethylcytosine is seen during olfactory neuron differentiation, and TET3 overexpression disrupts both the olfactory receptor expression pattern and the targeting of axons to the olfactory bulb (Colquitt et al, 2013). These two studies strongly suggest that the hydroxymethylation landscape is important for the correct formation of brain tissues, as already reported by Shi's group, which found TET3 to be required for normal development of the eye and brain in Xenopus laevis (Xu et al, 2012). Three recent papers have shed further light on the involvement of TET enzymes in brain development (Kaas et al, 2013; RudenKo et al, 2013; Zhang et al, 2013b). Zhang et al found that TET1 is needed for NPC proliferation and that in vivo depletion of TET1 affects neuron production in the hippocampus. Further characterization of the mice in Morris water maze experiments revealed that their short-term memory was reduced (Zhang et al, 2013b). Rudenko et al (2013) reached different conclusions: in fear conditioning and Morris water maze tests, they found a normal neuronal density in the hippocampus of TET1-knockout mice, with no short-term memory impairment but with a significant decrease in memory extinction (which affects long-term memory), possibly due to alteration of the long-term depression pathway. As these last results have been partially reproduced in mice overexpressing wild-type or mutant TET1 catalytic domains in the hippocampus, TET1 appears to act as an “inhibitor” of long-term memory, independently of its catalytic function (Kaas et al, 2013). While a consensus is hard to reach at this stage, it would seem that TETs affect both short- and long-term memory as well as NPC proliferation and differentiation. The discrepancies between Zhang's and Rudenko's results may be due to the use of different knockouts: Zhang et al targeted exons 11–13 of the TET1 protein, whereas Rudenko et al used knockout mice with a deleted exon 4 (previously described by Jaenisch and colleagues Rudenko et al, 2013; Zhang et al, 2013b). Further studies, including systematic conditional hippocampal knockout of TET1, TET2, and TET3 (and also double and triple knockouts), should help to assess these divergences and to distinguish the exact roles of the three TETs in neurogenesis.
Also beyond the neuronal context, reports show that the hydroxymethylcytosine pattern is highly dynamic during terminal cell differentiation, as described for preadipocytes, germ cells, and differentiating monocytes (Fujiki et al, 2013; Gan et al, 2013; Klug et al, 2013; de la Rica et al, 2013). During adipogenesis, the genome displays a global increase in 5hmC and local hydroxymethylation on genes that are later expressed in mature adipocytes (Fujiki et al, 2013). A similar initial global increase in 5hmC, followed by a global decrease, and the presence of differentially hydroxymethylated regions (dhMRs) on certain coding and non-coding genes are also observed during spermatogenesis (Gan et al, 2013). Finally, Klug et al (2013) and de la Rica et al (2013) have provided evidence that TETs, especially TET2, are essential to the proper differentiation of monocytes to osteoclasts: during this process, DNA demethylation preceded by 5hmC deposition is observed.
All in all, it thus seems that while 5hmC may decrease when mESCs lose their pluripotency, a global increase in this epigenetic mark can occur during terminal differentiation, sometimes followed by a subsequent decrease. This highlights the dynamicity of the hydroxymethylcytosine pattern and suggests a crucial role for the TET enzymes in cell differentiation and organ (e.g. brain) formation as well as a key role in cell reprogramming. With proteins so essential and so versatile, it comes as no surprise that their loss, or an alteration of the 5hmC pattern, can lead to diseases such as cancer or neurological disorders. Roles of altered TET functioning and hydroxymethylation in disease are discussed below.
TETs/modified cytosines in cancer and non-cancer diseases
TETs are on the one hand recognized as tumour suppressor and a hallmark of many cancers seems to be a significant decrease in 5hmC. This may be partly due to the well-known global hypomethylation that takes place during cell transformation, but in breast cancers, melanomas, and leukaemias without MLL rearrangements, reports have additionally revealed either a mutation in the TET2 gene (although the level of one TET does not always correlate with the global level of hydroxymethylcytosine) or substantial downregulation of all three TETs, for example by micro-RNAs (Tefferi et al, 2009; Haffner et al, 2011; Nestor et al, 2011; Hsu et al, 2012; Lian et al, 2012; Yang et al, 2012; Huang et al, 2013; Song et al, 2013a,b). In 2012, Hsu et al (2012) reported TET1 to be downregulated in prostate and breast cancer tissues, and found its loss to facilitate cell invasion in xenograft mouse models, at least via deregulation of “inhibitor of metalloproteinase” proteins (TIMPs). In human melanomas, which show a marked genome-wide decrease in hydroxymethylcytosine and more subtle changes in methylation patterns, the most decreased TET is TET2. When TET2 is overexpressed in human melanoma cells xenografted onto immunodeficient NSG mice, tumour growth is suppressed. This suggests a crucial involvement of TET2 and 5hmC in melanoma development (Lian et al, 2012). Furthermore, TET2 is often mutated in myeloid malignancies,. In 4–13% of myeloproliferative neoplasms and 20–25% of myelodysplastic syndromes, somatic deletions and TET2-inactivating mutations are found, but do not correlate clearly with prognosis (Tefferi et al, 2009; Bejar et al, 2011). TET2 mutations are also found in 7–23% of AMLs, where they correlate with poor prognosis. A major role of TET2 in leukaemia has been confirmed in TET2-knockout mice, where loss of the enzyme appears to increase the haematopoietic stem cell compartment and to skew cell differentiation towards the myeloid compartment, causing symptoms resembling those associated with TET2 mutations (Li et al, 2011; Moran-Crusio et al, 2011; Quivoron et al, 2011).
In fascinating contrast to the above, there are contexts where TETs appear to have an oncogenic action. They owe the designation “Ten-Eleven translocation enzyme” to a rare translocation of the histone methyl transferase gene MLL and of the TET1 coding sequence in acute myeloid leukaemia (AML), yielding a 5′ MLL-TET1 3′ chimera (Lorsbach et al, 2003). The role of this translocation in leukaemia is unclear, and it seems that oncogenic transformation driven by the rearranged MLL is observed with different translocation partners (de Boer et al, 2013). Yet Huang et al revealed in 2013 that TET1 plays an essential oncogenic role in MLL-rearranged leukaemia. It is aberrantly overexpressed in MLL-rearranged AML, while the TET2 and TET3 expression levels remain unchanged. ChIP-qPCR and expression experiments have further shown that the TET1 promoter is a direct target of MLL-fusion proteins and that the increase in TET1 leads to a global genomic increase of hydroxymethylcytosine. Furthermore, MLL fusions and TET1 both co-regulate genes such as HOXA9 or MEIS1 that inhibit apoptosis and enhance cell proliferation, causing transformation and leukaemogenesis (Huang et al, 2013).
In summary, the hydroxymethylcytosine level is often deregulated in cancer, but not always in the same manner: globally there may be an increase, as seen in MLL-rearranged leukaemia, or a marked decrease, as observed in many other cancer types. Recently, some elegant techniques have been created to precisely map 5hmC at single-nucleotide resolution (Booth et al, 2012; Yu et al, 2012). This could make this epigenetic modification an interesting marker for use in diagnosing cancers, especially blood cancers, for which cell samples can easily be obtained.
Growing attention is also focusing on the involvement of TET dysregulation in non-cancer diseases. There is increasing evidence that epigenetic dysfunction and resulting changes in gene expression may contribute at least partly to the aetiology of various pathologies, especially neurological disorders (see Table 1) (Jakovcevski & Akbarian, 2012). As mentioned above, the mammalian brain is very rich in hydroxymethylcytosine. In Rett syndrome, mutations in MeCP2 impair the binding of this protein to 5hmC, suggesting that the altered deposition of this mark by TET methyldioxygenases may also play a role in neurological diseases (Kriaucionis & Heintz, 2009; Mellen et al, 2012). Accordingly, several reports have revealed altered TET expression and an abnormal 5hmC pattern in the brains of patients suffering from psychosis, Huntington's disease, Friedreich's ataxia, and fragile X syndrome (Campuzano et al, 1996; Tassone et al, 2000; Saveliev et al, 2003; Sadri-Vakili & Cha, 2006; Dong et al, 2012; Guidotti et al, 2012; Al-Mahdawi et al, 2013; Auta et al, 2013; Chouliaras et al, 2013; Villar-Menendez et al, 2013; Wang et al, 2013; Yao et al, 2013; Coppieters et al, 2014). Psychosis is a broad category encompassing symptoms of schizophrenia and bipolar disorder. Psychotic patients have been found to display downregulated expression of several vulnerability genes, including GAD67, reelin, NR2A, and GAT1, in parallel with hypermethylation of the corresponding promoter regions (Dong et al, 2012). Recently, two groups additionally reported hyper-hydroxymethylation of the GAD67 promoter in the inferior parietal lobe, both in a psychotic cohort and in mice born with psychosis-like disorders (Dong et al, 2012; Matrisciano et al, 2012). To evaluate the possible role of 5hmC in GAD67 repression, it would be interesting to see if variation of the 5hmC level in GAD67 might correlate with TET binding to one or more corepressors such as SIN3A, and NurD. In addition, Dong et al (2012) revealed local variations in hydroxymethylcytosine content, a global increase in both TET1 and 5hmC in schizophrenic and bipolar patients, and persons suffering from depression displayed a similar, albeit insignificant trend. A TET1 increase has likewise been observed in psychotic patients with a history of alcohol abuse and interestingly, in lymphocytes of schizophrenic patients. The latter finding suggests that it might be possible to identify peripheral methylation/hydroxymethylation biomarkers for use in diagnosis/prognosis when a biopsy is not an option (Guidotti et al, 2012; Auta et al, 2013).
Table 1. This table summarizes the involvement of TETs and methylcytosine oxidation in neuronal diseases
|Rett syndrome||Decrease of MeCP2 binding on 5hmC|| |
Kriaucionis & Heintz (2009)
Mellen et al (2012)
|Psychosis||GAD67 promoter hyper-hydroxymethylation|| |
Dong et al (2012)
Matrisciano et al (2012)
Global increase of 5hmC
Increase of TET1 expression
Dong et al (2012)
Guidotti et al (2012)
Auta et al (2013)
|Huntington's disease||Decrease of 5hmC on ADORA2A promoter||Villar-Menendez et al (2013)|
Global decrease of 5hmC
Decrease of TET1 expression
|Wang et al (2013)|
|Friedreich's ataxia||Global increase of 5hmC||Al-Mahdawi et al (2013)|
|Fragile X syndrome|| |
Global decrease of 5hmC on gene bodies and promoters
Subtle increase of 5hmC on enhancers and repetitive elements
|Yao et al (2013)|
|Alzheimer's disease|| |
Global increase of 5hmC in hippocampus
Global decrease of 5hmC in temporal tissues
Coppieters et al (2014)
Chouliaras et al (2013)
Huntington's disease (HD) is a genetic disorder characterized by expansion of glutamine residues in the protein huntingtin, due to accumulation of the CAG triplet in the first exon of the corresponding gene. The symptoms include difficulties in motor coordination and also psychiatric disturbances, cognitive disorders, and weight loss (Sadri-Vakili & Cha, 2006). In 2013, Villar-Menendez et al (2013) showed that the promoter of the ADORA2A gene, encoding the G-protein coupled receptor A2AR known to be downregulated in HD and involved in the disease, displays a decreased 5hmC level in a mouse model of HD and in the putamens of HD patients. In contrast to the global increase in hydroxymethylcytosine found in psychosis, dot-blot experiments and analysis of 5hmC patterns by deep sequencing in an HD mouse model revealed a rather global decrease in 5hmC, accompanied by a significant decrease in TET1 expression. Analysis of the differentially hydroxymethylated regions further uncovered an alteration of canonical pathways involved in neuronal development and differentiation pathways. Hence, the loss of hydroxymethylcytosine may impair neuronal function in HD brains and appears as a novel epigenetic marker of the disease (Wang et al, 2013).
Friedreich's ataxia (FRDA) is another neurodegenerative disease due to expansion of a triplet (GAA). This expansion, here in the first intron of the FXN gene, leads to heterochromatin formation and gene silencing. Upstream from the repeats lay CpGs that appear hypermethylated in patients suffering from the disease. In the studies concerned, however, the techniques used to map 5mC could not distinguish this mark from 5hmC (Campuzano et al, 1996; Saveliev et al, 2003; Al-Mahdawi et al, 2013). In 2013, Al-Mahdawi et al (2013) addressed this issue, showing that nearly all the methylcytosine is in fact hydroxymethylcytosine in both FRDA and normal human cerebellar tissues, and that 5hmC is more abundant in the former than in the latter. They further hypothesized that the observed 5hmC increase might enhance production of FAST-1 antisense RNA, which could in turn mediate heterochromatin formation and FXN downregulation, causing the typical symptoms of the disease. Lastly, the fragile X genetic syndrome, triggered by FMR1 gene repression, is a widespread inherited cause of autism and mental retardation in boys (Tassone et al, 2000; Yao et al, 2013). While the disorder is known to be associated with hypermethylation of the FMR1 promoter and subsequent transcriptional gene silencing, possible global fragile-X-associated changes in DNA methylation/hydroxymethylation were not addressed until recently, when Yao et al reported a global decrease in 5hmC in gene bodies and promoters and a more subtle increase in cerebellum-specific enhancers and some repetitive elements. The same report also presents dhMR analyses highlighting changes affecting functional pathways in neuronal development. As these results were obtained from a knockin mouse model, it will be important to assess the global level of hydroxymethylcytosine in fragile X syndrome patients (Yao et al, 2013).
In each of the above-mentioned neuronal diseases, the global level of hydroxymethylation thus seems to be altered, either upward or downward according to the disease. Also, some disorders may involve several tissues in the brain, and both the global and specific 5hmC patterns may depend on the analysed tissue. This is exemplified by two recent studies showing that in Alzheimer's disease, the most common form of dementia in humans, hippocampal regions display a significant increase in 5hmC, while the middle frontal and temporal gyri show a significant decrease (Chouliaras et al, 2013; Coppieters et al, 2014).
In summary, the TET methyldioxygenase machinery seems altered in various diseases, notably cancers and neudegenerative disorders but also probably other important diseases such as oxidative-stress related disorders (see modes of direct regulation of TET enzymes). This strongly suggests that 5hmC patterns are important in many pathologies. As DNA can be recovered easily from tissues and is stable over time, 5hmC profiles might be used to find clinically relevant epigenetic biomarkers. Yet questions remain to be answered. It will notably be important to identify clearly the pathways affecting 5hmC profile changes and those affected by them. We also need to distinguish whether variations of the global level of this mark are causes or consequences of the diseases concerned. These important questions will surely keep a lot of laboratories excited and busy for the next few years.