TET Genes: new players in DNA demethylation and important determinants for stemness

Stem cells are defined as cells that have the ability to perpetuate themselves through self-renewal and to generate functional mature cells by differentiation. During each stage, coordinated gene expression is crucial to maintain the balance between self-renewal and differentiation. Disturbance of this accurately balanced system can lead to a variety of malignant disorders. In mammals, DNA cytosine-5 methylation is a well-studied epigenetic pathway that is catalyzed by DNA methyltransferases and is implicated in the control of balanced gene expression, but also in hematological malignancies. In this review, we focus on the TET (ten-eleven-translocation) genes, which recently were identified to catalyze the conversion of cytosine-5 methylation to 5-hydroxymethyl-cytosine, an intermediate form potentially involved in demethylation. In addition, members of the TET family are playing a role in ES cell maintenance and inner cell mass cell specification and were demonstrated to be involved in hematological malignancies. Recently, a correlation between low genomic 5-hydroxymethyl-cytosine and TET2 mutation status was shown in patients with myeloid malignancies. Stem cells are defined as cells that have the ability to perpetuate themselves through self-renewal and to generate functional mature cells by differentiation. During each stage, coordinated gene expression is crucial to maintain the balance between self-renewal and differentiation. Disturbance of this accurately balanced system can lead to a variety of malignant disorders. In mammals, DNA cytosine-5 methylation is a well-studied epigenetic pathway that is catalyzed by DNA methyltransferases and is implicated in the control of balanced gene expression, but also in hematological malignancies. In this review, we focus on the TET (ten-eleven-translocation) genes, which recently were identified to catalyze the conversion of cytosine-5 methylation to 5-hydroxymethyl-cytosine, an intermediate form potentially involved in demethylation. In addition, members of the TET family are playing a role in ES cell maintenance and inner cell mass cell specification and were demonstrated to be involved in hematological malignancies. Recently, a correlation between low genomic 5-hydroxymethyl-cytosine and TET2 mutation status was shown in patients with myeloid malignancies. Modification of nucleic acid bases without changing the primary DNA base sequence is observed in prokaryotic and eukaryotic cells, and it is responsible for a wide range of biological functions. The most common enzymatic DNA modification in these superkingdoms of life is methylation of the 5-position of cytosine. In mammalian cells, enzymatic modification within CpG islands can act as a stably inherited modification affecting gene regulation, cellular differentiation, genomic imprinting, X-inactivation, and embryogenesis [1Holliday R. Pugh J.E. DNA modification mechanisms and gene activity during development.Science. 1975; 187: 226-232Crossref PubMed Scopus (1431) Google Scholar, 2Okano M. Bell D.W. Haber D.A. Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4682) Google Scholar, 3Reik W. Dean W. Walter J. Epigenetic reprogramming in mammalian development.Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2497) Google Scholar, 4Riggs A.D. X inactivation, differentiation, and DNA methylation.Cytogenet Cell Genet. 1975; 14: 9-25Crossref PubMed Scopus (939) Google Scholar]. Base modification is reversible in cells and is also required to maintain the genomic integrity of cellular DNA and suppression of transposable elements to protect from mutagenic effects [5Jaenisch R. Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.Nat Genet. 2003; 33: 245-254Crossref PubMed Scopus (4849) Google Scholar]. Recently, non-CpG context cytosine methylation was found in embryonic stem cells but not in differentiated cells. Interestingly, in IMR90 fetal lung fibroblast-induced pluripotent stem (iPS) cells, restoration of non-CpG methylation was observed, suggesting that different methylation mechanisms are involved in embryonic stem cells (ESCs) for maintenance of a pluripotent state [6Lister R. Pelizzola M. Dowen R.H. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3499) Google Scholar]. DNA modifications other than methylation, such as 5-hydroxymethylpyrimidines, are known from T-even phages used in countering a host DNA response. Further, the formation of β-d-glucosyl hydroxymethyl-uracil (base J) by oxidation of the methyl group on thymine leading to the formation of 5-hydroxymethyuracil (5-hmU) was found in the eukaryotic parasites Trypanosomes and Leishmania [7Cliffe L.J. Kieft R. Southern T. et al.JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes.Nucleic Acids Res. 2009; 37: 1452-1462Crossref PubMed Scopus (68) Google Scholar, 8Yu Z. Genest P.A. ter Riet B. et al.The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase.Nucleic Acids Res. 2007; 35: 2107-2115Crossref PubMed Scopus (73) Google Scholar]. Recently, 5-hydroxymethyl-cytosine (5hmC) was identified as a new epigenetic marker in mouse purkinje neurons and ESCs [9Kriaucionis S. Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.Science. 2009; 324: 929-930Crossref PubMed Scopus (2191) Google Scholar, 10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar]. In principle, alterations in the specific methylation status of promoter regions or imprinted genes can lead to activation of normally inactive oncogenes, or the inactivation of genes that suppress tumorigenesis [11Chuang J.C. Jones P.A. Epigenetics and microRNAs.Pediatr Res. 2007; 61: 24R-29RCrossref PubMed Scopus (552) Google Scholar, 12Jost E. Galm O. EHA scientific workshop report: the role of epigenetics in hematological malignancies.Epigenetics. 2007; 2: 71-79Crossref PubMed Scopus (10) Google Scholar]. Specifically the activation of developmental genes and inactivation of DNA repair systems can lead to cancer of various types. In acute myeloid leukemia (AML), which is characterized by an impaired differentiation and enhanced proliferation resulting in the growth of a clonal population of immature hematologic blasts, a pattern of dysregulated methylation can be observed and distinct biological subgroups of AML can be identified and correlated to patient survival [13Figueroa M.E. Lugthart S. Li Y. et al.DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia.Cancer Cell. 2010; 17: 13-27Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar]. The cause for this tremendous dysregulation of methylation pattern remains unknown, but recently ten-eleven-translocation (TET)1, a fusion partner of the mixed lineage leukemia (MLL) gene in the translocation t(10;11)(q22;q23) [14Burmeister T. Meyer C. Schwartz S. et al.The MLL recombinome of adult CD10-negative B-cell precursor acute lymphoblastic leukemia: results from the GMALL study group.Blood. 2009; 113: 4011-4015Crossref PubMed Scopus (78) Google Scholar, 15Lorsbach R.B. Moore J. Mathew S. Raimondi S.C. Mukatira S.T. Downing J.R. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23).Leukemia. 2003; 17: 637-641Crossref PubMed Scopus (330) Google Scholar, 16Ono R. Taki T. Taketani T. Taniwaki M. Kobayashi H. Hayashi Y. LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23).Cancer Res. 2002; 62: 4075-4080PubMed Google Scholar], was identified to catalyze the conversion of cytosine-5 methylation (5mC) to 5hmC, an intermediate form possibly involved in DNA demethylation [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar]. Furthermore, Tet1 has been shown to play a role in ES cell maintenance and inner cell mass specification [17Ito S. D'Alessio A.C. Taranova O.V. Hong K. Sowers L.C. Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.Nature. 2010; 26 (466(7310):1129–1133)Google Scholar]. Additionally, TET2, a structural homologue of TET1 and member of the TET family, was found to be frequently mutated in myeloid malignancies such as myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN), which are both able to progress to sAML as well as AML. The TET family consists of three members, namely TET1, TET2, and TET3. TET1 is located on 10q21 and contains 12 exons spanning a sequence of 134 kb. The mRNA has a length of 9.6 kb, while the coding sequence consists of about 6.4 kb coding for a protein of 2136 amino acids (AA) [18Abdel-Wahab O. Mullally A. Hedvat C. et al.Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.Blood. 2009; 114: 144-147Crossref PubMed Scopus (586) Google Scholar]. TET2 is located on 4q24 and spans 96 kb coding for an mRNA with the size of 9.7 kb and a coding sequence of 6 kb, although translation results in a protein of 2002 AA [19Hussein K. Abdel-Wahab O. Lasho T.L. et al.Cytogenetic correlates of TET2 mutations in 199 patients with myeloproliferative neoplasms.Am J Hematol. 2010; 85: 81-83Crossref PubMed Scopus (21) Google Scholar]. The TET2 gene contains 11 exons [20Strausberg R.L. Feingold E.A. Grouse L.H. et al.Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.Proc Natl Acad Sci U S A. 2002; 99: 16899-16903Crossref PubMed Scopus (1577) Google Scholar], and the resulting messenger RNA (mRNA) can be subjected to three different isoforms due to alternative splicing. One isoform lacks exon 3b, resulting in 2002 (AA), the second isoform is missing all exons after exon 3c, resulting in a stop codon at exon 3b, which leads to the length of 1164 AA, and the third isoform is lacking exon 3b and 3c, which forms a protein with 1194 AA. The three isoforms show different expression in various body tissues, including hematologic lineages [21Langemeijer S.M.C. Kuiper R.P. Berends M. et al.Acquired mutations in TET2 are common in myelodysplastic syndromes.Nat Genet. 2009; 41: 838-842Crossref PubMed Scopus (639) Google Scholar]. TET3 is located on 2p13, with a relatively small size of 61.8 kb at the genomic level, coding for a 10.9-kb mRNA and a coding sequence of 4.9 kb [18Abdel-Wahab O. Mullally A. Hedvat C. et al.Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.Blood. 2009; 114: 144-147Crossref PubMed Scopus (586) Google Scholar]. The resulting protein consists of 1660 AA. TET3 contains 9 exons, and 3 putative isoforms have been reported by complementary DNA screening (full length isoform 1) [22Gerhard D.S. Wagner L. Feingold E.A. et al.The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).Genome Res. 2004; 14: 2121-2127Crossref PubMed Scopus (434) Google Scholar], isoform 2 missing AA 1440–1555 [23Ishikawa K. Nagase T. Nakajima D. et al.Prediction of the coding sequences of unidentified human genes. VIII. 78 new cDNA clones from brain which code for large proteins in vitro.DNA Res. 1997; 4: 307-313Crossref PubMed Scopus (79) Google Scholar], and isoform 3 missing AA 728-1660, but no expression data of the 3 isoforms has been shown so far. All TET family members contain three metal binding sites (TET1: AA 1672, 1674, 2028; TET2: 1382, 1384, 1884; TET3: 942, 944, 1538) which enable the proteins to bind Fe(II)-ions and thus render it catalytically active. The binding of 2-oxoglutarate is mediated by one AA in each TET family member (TET1: AA 2043, TET2: AA 1896, TET3: AA 1553). In addition to the zinc finger domain, TET1 contains three nuclear localization signals (NLS) (AA 20-50, 620–653, 1158–1162), which indicate a potential function of TET1 in the nucleus [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar]. A further analysis of TET2 and TET3 using PredictNLS [24Cokol M. Nair R. Rost B. Finding nuclear localization signals.EMBO Rep. 2000; 1: 411-415Crossref PubMed Scopus (564) Google Scholar] does not reveal the presence of any NLS. Secondary structure predictions for TET1 indicate an N-terminal α-helix followed by a cysteine-rich domain (CD) and several β-strands, which are forming a 2-oxoglutarate (2OG)-Fe(II) oxygenase characteristic double-stranded β-helix (DSBH). In case of TET1, the cysteine-rich region plus DSBH was confirmed as catalytic domain [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar]. Additionally, TET1 has a functional binuclear Zn-chelating CXXC-domain located N-terminally to the 2OG-Fe(II) oxygenase domain. This CXXC-domain also occurs in several chromatin proteins, e.g., DNA methyltransferase 1 and the DNA-binding protein MBD1 [25Iyer L.M. Tahiliani M. Rao A. Aravind L. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.Cell Cycle. 2009; 8: 1698-1710Crossref PubMed Scopus (311) Google Scholar]. The CXXC domain seems to be absent in TET3 and TET2, but a closer look at the chromosomal environment of TET2 reveals the gene CXXC4, which could indicate a chromosomal inversion of the CXXC-domain of TET2 and a subsequent translocation. Yet, it is speculated that interaction of TET2 and CXXC4 leads to the correct function of TET2 [25Iyer L.M. Tahiliani M. Rao A. Aravind L. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.Cell Cycle. 2009; 8: 1698-1710Crossref PubMed Scopus (311) Google Scholar]. When the protein sequences of the TET family members are aligned to each other, three homology peaks are observable: one spanning from the CD into the DSBH and two peaks at the end of the DSBH. This suggests an important function for these particular domains in substrate binding and enzymatic activity (Fig. 1). In early embryogenesis, CpG methylation is essential for X-inactivation and asymmetric expression of imprinted genes [26Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development.Nature. 2007; 447: 425-432Crossref PubMed Scopus (1562) Google Scholar]. Methylation of CpG islands in somatic cells shows a general correlation with gene expression. CpG-DNA modifications other than methylation are primarily known from caudate bacteriophages, which modify bases such as 5-hydroxymethylpyrimidines and their mono- or diglycosylated derivatives, and N6-carbamoylmethyl adenines to counter host DNA restriction responses [27Warren R.A. Modified bases in bacteriophage DNAs.Annu Rev Microbiol. 1980; 34: 137-158Crossref PubMed Scopus (175) Google Scholar]. Another well-studied eukaryotic modification is the formation of β-d-glucosyl-hydroxymethyluracil or base J from thymine in Euglenozoa. Recent bioinformatic work from Tahiliani et al. [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar] led to the prediction of the three human enzymes TET1-3 as Fe(II) and 2OG oxygenases that catalyze 5-methylcytosine hydroxylation (Fig. 2). TET family members were found to be homologues of the 2OG-Fe(II) oxygenases JBP1 and JBP2, which catalyze the conversion of thymine to β-d-glucosyl hydroxymethyl-uracil in Trypanosomes. This process generates the base J, which is present in silenced copies of genes [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar, 28Borst P. Sabatini R. Base J: discovery, biosynthesis, and possible functions.Annu Rev Microbiol. 2008; 62: 235-251Crossref PubMed Scopus (140) Google Scholar]. The close relationship of JBP and TET genes leads to the assumption that also members of the TET family members are catalytically active 2OG-Fe(II) oxygenases. Human 2OG oxygenases are involved in a diverse range of biological functions, including histone demethylation, collagen stabilization, DNA repair, hypoxia sensing, and fatty acid metabolism [29Loenarz C. Schofield C.J. Expanding chemical biology of 2-oxoglutarate oxygenases.Nat Chem Biol. 2008; 4: 152-156Crossref PubMed Scopus (407) Google Scholar]. Overexpression of TET1 in HEK293T cells resulted in reduced 5mC levels, while this effect was not observed with overexpression of mutated TET1, which impairs the predicted Fe(II) binding sites, suggesting that the catalytic activity of TET1 is dependent on Fe(II)-binding. Furthermore, RNA interference−mediated depletion of endogenous TET1 resulted in an about 40% decrease in 5hmC levels in ESCs [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar]. However, thymine conversion to hydroxymethyluracil (hmU) was not detectable, suggesting TET1 may be specific for 5mC. The enzymatic activity of Tet1 is conserved in human and mouse. Overexpression of both murine Tet1 and Tet2 could reduce global 5mC staining in both U2OS and HEK293T cells. Interestingly, Tet3 displayed weak enzymatic activity in these cells [17Ito S. D'Alessio A.C. Taranova O.V. Hong K. Sowers L.C. Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.Nature. 2010; 26 (466(7310):1129–1133)Google Scholar]. This recent study also strongly supports the hypothesis that TET1 and potentially other TET family members are responsible for 5hmC generation in ESCs under physiological conditions. Identifying the functional roles of hmC at cellular and physiological levels is an important objective. A recent study reports the direct reversible conversion of hmC within double-stranded DNA to unmodified cytosine and free formaldehyde, via covalent catalysis by a bacterial DNA-methyltransferase (DNMT) lacking cofactors [30Liutkeviciute Z. Lukinavicius G. Masevicius V. Daujotyte D. Klimasauskas S. Cytosine-5-methltransferases add aldehydes to DNA.Nat Chem Biol. 2009; 5: 400-402Crossref PubMed Scopus (113) Google Scholar]. This suggests that the modification of 5mC to 5hmC might play a crucial role in gene regulation. Recently, the expression of Tet1 and Tet2 was shown in murine ES cells (E14). Interestingly, Tet3, which is highly expressed in most of the organs including human hematopoietic stem cells and murine V6.5 ES cells, is not expressed in murine E14 ES cells [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar, 17Ito S. D'Alessio A.C. Taranova O.V. Hong K. Sowers L.C. Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.Nature. 2010; 26 (466(7310):1129–1133)Google Scholar]. The Zhang group could demonstrate with their findings that knockdown of Tet1 in ES cells via shRNA results in morphological abnormalities and a decrease of alkaline phosphatase activity. Furthermore, a reduced cell growth was observed, which was not correlating to an increase in apoptosis but rather to a self-renewal defect, which resulted in a reduction of the colony forming capacity and indicated a decrease in stemness of these cells. Additionally, Tet1 depletion induced a reduction of Nanog expression and a minor decrease of Oct4 and Sox2 expression (see Fig. 3). Using ES cell surface marker staining, the group showed that knockdown of Tet1 results in a 10–15% increase in Stage-Specific Embryonic Antigen−negative cells. Upon induced differentiation by withdrawal of leukemia inhibitory factor or retinoic-acid treatment, Tet1 expression was greatly downregulated on both mRNA and protein level. The authors attributed these findings to the function of Tet1 in stem cell maintenance. This was further supported by the observation that Tet1 knockdown induced upregulation of Cdx2, Hand1, Gata6, and Gata4. In addition, reduced expression of Tet1 results in positive staining for markers of either the trophectoderm lineage or the primitive endoderm under these conditions. During embryogenesis, the group could detect high expression of Tet1 in the inner cell mass, small interfering RNA−mediated knockdown of Tet1 in single cells at the two-cell stage resulted in embryonic cell specification toward trophectoderm lineage. The role of Tet1 for stemness in ES cells was further underlined by the assumption that Tet1 is directly interacting with the Nanog promoter, thereby preventing hypermethylation of the promoter (see Fig. 3). Exogenous expression of Nanog was able to rescue the morphological abnormalities and alkaline-phosphatase activity, which both was observed in Tet1 knockdown cells. The knockdown of Tet2 did not show obvious effects in ES cells. All three TET genes showed broad expression pattern in different tissues, they are abundantly expressed in most of the normal hematopoietic cells. In contrast to TET1, TET2 and TET3 are higher expressed in hematopoietic cells [21Langemeijer S.M.C. Kuiper R.P. Berends M. et al.Acquired mutations in TET2 are common in myelodysplastic syndromes.Nat Genet. 2009; 41: 838-842Crossref PubMed Scopus (639) Google Scholar, 31Langemeijer S.M. Aslanyan M.G. Jansen J.H. TET proteins in malignant hematopoiesis.Cell Cycle. 2009; 8: 4044-4048Crossref PubMed Scopus (52) Google Scholar]. Among the hematopoietic subpopulations expression of TET2 and TET3 are highest in granulocytes. Induction of granulocytic differentiation in the promyelocytic cell line NB4 showed upregulation of TET2 expression [21Langemeijer S.M.C. Kuiper R.P. Berends M. et al.Acquired mutations in TET2 are common in myelodysplastic syndromes.Nat Genet. 2009; 41: 838-842Crossref PubMed Scopus (639) Google Scholar]. Furthermore, all aforementioned TET2 isoforms were found to be expressed in most of the analyzed tissues. In a recent analysis, Ko et al. [32Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1069) Google Scholar] have shown that Tet genes are expressed in LSK (Lin− Sca-1+ c-Kithi) containing hematopoietic stem cells and their expression was progressively downregulated as LSK differentiated into progenitors [32Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1069) Google Scholar]. Tet2 mRNA was shown to be expressed in common lymphoid progenitors, its expression gradually increased in early B-cell precursors in the bone marrow then declined slightly in mature B cells. In contrast, it was progressively downregulated during early T-cell development [32Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1069) Google Scholar]. Furthermore, depletion of Tet2 mRNA by short hairpin RNA in murine bone marrow stem/progenitor cells promoted an expansion of monocyte/macrophage cells in the presence of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation with macrophage colony-stimulating factor (M-CSF) did not lead to an expansion of monocyte/macrophage cells in Tet2-depleted bone marrow/progenitor cells [32Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1069) Google Scholar]. In these experiments, no increase in short-term proliferation of Tet2-depleted cells was detected by pulse-labeling with bromodeoxyuridine, which is incorporated in the DNA during replication and thus a marker for proliferation. This study indicates that Tet2 is important for normal myelopoiesis [32Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1069) Google Scholar]. In 2002, the TET1 gene, previously called LCX (leukemia-associated protein with a CXXC domain), had been identified as a fusion partner of the mixed lineage leukemia (MLL) gene in an adult AML patient with translocation t(10;11)(q22;q23). Later, a t(10;11) translocation was also found in a pediatric AML patients and patients with acute lymphoblastic leukemia [14Burmeister T. Meyer C. Schwartz S. et al.The MLL recombinome of adult CD10-negative B-cell precursor acute lymphoblastic leukemia: results from the GMALL study group.Blood. 2009; 113: 4011-4015Crossref PubMed Scopus (78) Google Scholar, 15Lorsbach R.B. Moore J. Mathew S. Raimondi S.C. Mukatira S.T. Downing J.R. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23).Leukemia. 2003; 17: 637-641Crossref PubMed Scopus (330) Google Scholar, 16Ono R. Taki T. Taketani T. Taniwaki M. Kobayashi H. Hayashi Y. LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23).Cancer Res. 2002; 62: 4075-4080PubMed Google Scholar]. Translocations, which create fusion genes with MLL, are associated with truncation of MLL and often predict a poor prognosis in acute lymphoblastic leukemia [33Holleman A. Cheok M.H. den Boer M.L. et al.Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.N Engl J Med. 2004; 351: 533-542Crossref PubMed Scopus (528) Google Scholar]. In the case of MLL-TET1, exon 6 of MLL is fused to the C-terminal part (exon 8, 9, or 11) of TET1, which contains the catalytical domain. The fusion protein results in loss of the CXXC domain and all three NLS of TET1 and thus subsequently may influence DNA binding and nuclear localization of TET1 [14Burmeister T. Meyer C. Schwartz S. et al.The MLL recombinome of adult CD10-negative B-cell precursor acute lymphoblastic leukemia: results from the GMALL study group.Blood. 2009; 113: 4011-4015Crossref PubMed Scopus (78) Google Scholar, 15Lorsbach R.B. Moore J. Mathew S. Raimondi S.C. Mukatira S.T. Downing J.R. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23).Leukemia. 2003; 17: 637-641Crossref PubMed Scopus (330) Google Scholar, 16Ono R. Taki T. Taketani T. Taniwaki M. Kobayashi H. Hayashi Y. LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23).Cancer Res. 2002; 62: 4075-4080PubMed Google Scholar]. However, the CXXC domain of MLL is retained, which may still enable the truncated form of MLL to bind to CpG motifs. Until now, it was not known if the transforming potential was due to the MLL part of the fusion protein, or if potential maintenance or tumor-suppressor activity of TET1 was disrupted. Recently, a study has shown that fusion proteins of MLL-AF9 result in increased demethylation of Hoxa9, while a disruption of the CXXC domain of MLL in this fusion protein resulted in regular methylation [34Cierpicki T. Risner L.E. Grembecka J. et al.Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia.Nat Struct Mol Biol. 2010; 17: 62-68Crossref PubMed Scopus (142) Google Scholar]. However, Tahiliani et al. [10Tahiliani M. Koh K.P. Shen Y. et al.Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4452) Google Scholar] suggest a loss of function (LOF) of TET1 as transforming factor. In contrast, no somatic mutations of TET1 have been found in MPN, chronic myelomonocytic leukemia, or AML so far [18Abdel-Wahab O. Mullally A. Hedvat C. et al.Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.Blood. 2009; 114: 144-147Crossref PubMed Scopus (586) Google Scholar]. Among all three TET family members, TET2 is most frequently mutated in myeloid malignancies. With an overall mutation rate of 19.5%, the frequency of mutations varies in the different disease entities. In de novo AML, TET2 is mutated in 12.1% of the cases [18Abdel-Wahab O. Mullally A. Hedvat C. et al.Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.Blood. 2009; 114: 144-147Crossref PubMed Scopus (586) Google Scholar]. In MPN, the rate of mutation is 14.1% [19Hussein K. Abdel-Wahab O. Lasho T.L. et al.Cytogenetic correlates of TET2 mutations in 199 patients with myeloproliferative neoplasms.Am J Hematol. 2010; 85: 81-83Crossref PubMed Scopus (21) Google Scholar, 35Colaizzo D. Tiscia G.L. Pisanelli D. et al.New TET2 gene mutations in patients with myeloproliferative