Leukemia (2014) 28, 485–496 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu SPOTLIGHT REVIEW The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases E Solary1,2,3, OA Bernard1,3,4, A Tefferi5, F Fuks6 and W Vainchenker1,2,3 Ten-Eleven Translocation-2 (TET2) inactivation through loss-of-function mutation, deletion and IDH1/2 (Isocitrate Dehydrogenase 1 and 2) gene mutation is a common event in myeloid and lymphoid malignancies. TET2 gene mutations similar to those observed in myeloid and lymphoid malignancies also accumulate with age in otherwise healthy subjects with clonal hematopoiesis. TET2 is one of the three proteins of the TET (Ten-Eleven Translocation) family, which are evolutionarily conserved dioxygenases that catalyze the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and promote DNA demethylation. TET dioxygenases require 2-oxoglutarate, oxygen and Fe(II) for their activity, which is enhanced in the presence of ascorbic acid. TET2 is the most expressed TET gene in the hematopoietic tissue, especially in hematopoietic stem cells. In addition to their hydroxylase activity, TET proteins recruit the O-linked b-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) enzyme to chromatin, which promotes post-transcriptional modifications of histones and facilitates gene expression. The TET2 level is regulated by interaction with IDAX, originating from TET2 gene fission during evolution, and by the microRNA miR-22. TET2 has pleiotropic roles during hematopoiesis, including stem-cell self-renewal, lineage commitment and terminal differentiation of monocytes. Analysis of Tet2 knockout mice, which are viable and fertile, demonstrated that Tet2 functions as a tumor suppressor whose haploinsufficiency initiates myeloid and lymphoid transformations. This review summarizes the recently identified TET2 physiological and pathological functions and discusses how this knowledge influences our therapeutic approaches in hematological malignancies and possibly other tumor types. Leukemia (2014) 28, 485–496; doi:10.1038/leu.2013.337 Keywords: TET2; epigenetics; DNA methylation; dioxygenase; stem cell; differentiation INTRODUCTION zinc-finger domain and a carboxy-terminal catalytic Fe(II)- and Five years ago, the demonstration that monoallelic or biallelic loss- a-ketoglutarate (a-KG)-dependent dioxygenase domain inserted of-function mutations and deletions recurrently target the TET2 in a cystein-rich domain. In jawed vertebrates, the TET genes (Ten-Eleven Translocation 2) gene in myeloid malignancies,1,2 and underwent triplication. TET1 and TET3 have also a CXXC domain, the simultaneous demonstration that proteins of the TET family whereas a chromosomal inversion during vertebrate evolution have a key role in the conversion of 5-methyl-cytosine (5-mC) to split the third TET gene into distinct segments encoding the 5-hydroxymethyl-cytosine (5-hmC),3 have opened a large field of catalytic domain (the TET2 gene) and the DNA-binding CXXC investigation, both in basic science and clinics. The present review domain (the CXXC4/IDAX gene). The latter is transcribed in the opposite direction and the protein exerts a regulatory function on summarizes our current understanding of the protein functions SPOTLIGHT 6 and regulation and discusses the significance of its deregulation in TET2 level expression. The CXXC motif may be responsible for 6,7 hematological malignancies as well as potential therapeutic direct (TET1 and TET3) or indirect (TET2) DNA binding. Different opportunities that emerge from these studies. TET enzymes exhibit distinct expression patterns in vivo, with TET1 being mainly expressed in embryonic stem cells. TET2 and TET3 are more ubiquitous, with TET2 expression predominating in a variety of differentiated tissues, especially in hematopoietic and THE TET FAMILY OF ENZYMES neuronal lineages.3 The three enzymes of the TET family (TET1, TET2 and TET3) identified in humans are evolutionarily conserved dioxygenases (Figure 1a). The TET1 gene was initially described as a fusion partner of the MLL (myeloid/lymphoid or mixed lineage leukemia) a-KG DEPENDENCY OF TET ENZYMES LINKS METABOLISM TO gene in an acute myeloid leukemia (AML) with a t(10;11)(q22;q23) EPIGENETICS translocation, with TET2 and TET3 being identified by homology TET enzymes are one of the homeostatic links between searches.4,5 It appeared that most animals had a single TET intracellular metabolism and epigenetic gene regulation.8 Like a orthologue, characterized by an amino-terminal CXXC-type number of chromatin-modifying enzymes, such as the JmjC 1Hematology Department, Gustave Roussy, Villejuif, France; 2Inserm UMR1009, Gustave Roussy, Villejuif cedex, France; 3Faculty of Medicine, University Paris-Sud, Le Kremlin- Biceˆtre, France; 4Inserm UMR985, Gustave Roussy, Villejuif, France; 5Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA and 6Faculty of Medicine, Laboratory of Cancer Epigenetics, Universite´ Libre de Bruxelles, Brussels, Belgium. Correspondence: Professor E Solary, Inserm UMR1009, Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif cedex, France. E-mail: [email protected] Received 11 October 2013; accepted 14 October 2013; accepted article preview online 13 November 2013; advance online publication, 6 December 2013 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 486 a 1 1412/1589 1620/2138 TET1 1 28/1299 1322/2002 TET2 1 689/859 882/1660 TET3 b Chromosome 4q24 106,067,032 TET2 106,200,960 106,389,463 IDAX 105,416,058 c Exon 3 4 5 6 7 8 9 10 11 Figure 1. TET proteins and the TET2 gene. (a) Primary structure of TET proteins. The CXXC domain of TET1 and TET3 is indicated in red, the cystein-rich domains of the three proteins are in gray and the double-stranded b-helix 2-oxoglutarate and Fe(II)-dependent dioxygenase SPOTLIGHT domains are in blue. Each of these proteins contains three Fer-binding domains and one site for 2-oxoglutarate binding in the dioxygenase domain (not shown). (b) The IDAX gene, also known as CXXC4, originates from the ancestral TET2 gene fission during vertebrate evolution and is transcribed in the opposite direction. (c) Five distinct models of Tet2 deletion in the mouse have been established. Each arrow indicates the targeted part of the gene in these models (the mouse phenotypes are described in Table 2). domain-containing histone demethylases, TET dioxygenases example, in mouse embryonic stem cells (ESCs), there are 45 require a-KG (also known as 2-oxoglutarate), oxygen and Fe(II) 5-hmCs per 1000 5-mC.14,15 for their activity, which is enhanced in the presence of ascorbic 5-hmC is a dynamic epigenetic state of DNA and the conversion acid.9,10 Fe(II), 2OG-dependent dioxygenases have a common of 5-mC into 5-hmC initiates demethylation that can occur in structural platform.11 Exons 7–11 of the TET2 gene encode a core several ways16,17 (Figure 3). First, because 5-hmC may not be made of eight anti-parallel b-strands folded into a ‘Jelly-roll’ motif recognized by DNA methyltransferase 1, the oxidation of 5-mC that harbors the active site. The so-called ‘2-His-1-carboxylate into 5-hmC may favor a passive demethylation that is DNA triad’, made of three residues in the active site (two histidine and replication-dependent.17 Secondly, 5-hmC could be converted by one aspartate or glutamate residues), forms a Fe(II)-binding the activation-induced deaminase/apolipoprotein B mRNA-editing platform. The Fe(II) metal center, locked in this triad, binds enzyme complex family of cytosine demethylases into 2-oxoglutarate and O2 on the other side. a-KG, which can be 5-hydroxymethyluracil (5-hmU), to be repaired by DNA glycosylases derived from several sources including isocitrate and glutamic and the base-excision repair pathway.18,19 The physiological acid, is decarboxylated to succinate during the oxidation reaction.8 importance of this second pathway in mammal cells remains Somatic mutations in isocitrate dehydrogenase (IDH) enzymes, controversial. Third, iterative oxidation of 5-mC and 5-hmC by TET either cytosolic IDH1 or mitochondrial IDH2, which are observed enzymes generate 5-formylcytosine and 5-carboxylcytosine, which in various tumors including myeloid malignancies,12 unmask a are recognized and excised by thymine DNA glycosylase into an latent ability of these enzymes to produce the R enantiomer abasic site. Subsequent repair by the base-excision repair pathway of 2-hydroxyglutarate, an ‘oncometabolite’ that inhibits restores an unmodified cytosine.19–22 2-oxoglutarate-dependent enzymes, including TET dioxygenases The dynamic methylation/demethylation cycle that involves TET (Figure 2). Potentially, TET enzymes may be also sensitive to and thymine DNA glycosylase enzymes (TET/thymine DNA changes in oxygen availability and susceptible to reactive oxygen glycosylase cycle) was shown recently to occur at a large number species and carcinogenic metals that displace iron such as arsenic, of genomic loci across the genome.23 Interestingly, long-lived nickel or chromium.13 5-hmC, and to a lesser extent, short-lived 5-formylcytosine and 5-carboxylcytosine may have also DNA demethylation- independent functions and serve as stable epigenetic marks that recruit specific readers, DNA repair proteins and transcription TET ENZYMES PROMOTE DNA DEMETHYLATION factors, with limited overlap between these proteins.24–27 Methylation at carbon atom 5 of the nucleotide cytosine In addition, most 5-mC-binding