The methylation status of DNA influences many biological processes during mammalian development and is known to be highly aberrant in cancer. In mammalian cells, DNA methylation occurs primarily as symmetrical methylation of cytosine in the context of the dinucleotide CpG, and the presence of high levels of 5-methyl- cytosine (5mC) at promoters is generally correlated with diminished gene expression. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) in DNA. As a result, TET proteins alter DNA methylation status in a novel and hitherto unprecedented way. Simultaneously, several labs reported that TET2 mutations are frequently associated with myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and myeloid malignancies such as chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). Two other genes frequently mutated in these patients include those encoding the DNA methyltransferase DNMT3A and the polycomb group protein ASXL1, a component of a complex that deubiquitinates H2A. In this proposal we will explore, at a molecular level using appropriate mouse models, the roles of Tet2 and Tet3 in hematopoiesis and myeloid function (Aim 1).
In Aims 2 and 3, we will investigate the relation of Tet2/ Tet3 to Dnmt3a and Asxl1 respectively. We have developed many methods and generated many reagents relevant to these proposed studies, including quantitative methods to measure overall genomic levels of 5hmC in bone marrow samples from patients with MDS/ MPN/ CMML/ secondary AML;and innovative strategies for mapping the genomic location of 5hmC and profiling 5hmC at single-base resolution. We have generated mice with conditional disruption of the Tet2 and Tet3 genes;and have uncovered a novel relation between 5hmC and the polycomb complex. WE have shown in ES cells that 5hmC is present predominantly at the promoters of genes that are (i) inactive but """"""""poised"""""""" to be expressed upon ES cell differentiation;(ii) bear dual (""""""""bivalent"""""""") H3K4me3 and H3K27me3 marks;and (iii) are bound by components of the polycomb complexes PRC1 and PRC2 (the H3K27me3 mark is deposited by PRC2). By defining the genome-wide changes in DNA methylation, DNA hydroxymethylation and histone modifications that occur as a result of loss of function of Tet2 and Tet3 and selected leukemia-associated mutations in Dnmt3a and Asxl1, our proposed studies will provide fundamental insights into how these proteins control chromatin structure and the epigenetic landscape at their target genes. This information will help us understand how changes that occur as a result of somatic mutations in these gene products in stem cells might predispose to myeloid cancers in humans.

Public Health Relevance

In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a very minor base known as 5-methylcytosine (5mC) that has a disproportionately crucial role. This fifth base is produced by attaching a methyl group to one of the major bases, cytosine (C). We recently identified a new class of proteins known as TET proteins that convert 5-methylcytosine to the sixth base, 5- hydroxymethylcytosine (5hmC). The function of one of the TET proteins, TET2, is lost, and 5hmC levels are decreased, in a variety of precancerous states and cancers involving specific classes of blood cells in humans. In this proposal we plan to investigate the role of TET proteins and 5hmC in these cancers of the blood.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cancer Molecular Pathobiology Study Section (CAMP)
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Mufson, R Allan
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La Jolla Institute
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Zang, Shengbing; Li, Jia; Yang, Haiyan et al. (2017) Mutations in 5-methylcytosine oxidase TET2 and RhoA cooperatively disrupt T cell homeostasis. J Clin Invest 127:2998-3012
Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini et al. (2017) TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells. Nat Immunol 18:45-53
Tsagaratou, Ageliki; Lio, Chan-Wang J; Yue, Xiaojing et al. (2017) TET Methylcytosine Oxidases in T Cell and B Cell Development and Function. Front Immunol 8:220
Lio, Chan-Wang; Zhang, Jiayuan; González-Avalos, Edahí et al. (2016) Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility. Elife 5:
Li, Xiang; Yue, Xiaojing; Pastor, William A et al. (2016) Tet proteins influence the balance between neuroectodermal and mesodermal fate choice by inhibiting Wnt signaling. Proc Natl Acad Sci U S A 113:E8267-E8276
Äijö, Tarmo; Huang, Yun; Mannerström, Henrik et al. (2016) A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways. Genome Biol 17:49
Montagner, Sara; Leoni, Cristina; Emming, Stefan et al. (2016) TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities. Cell Rep 15:1566-1579
Zhang, Xiaotian; Su, Jianzhong; Jeong, Mira et al. (2016) DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells. Nat Genet 48:1014-23
Äijö, Tarmo; Yue, Xiaojing; Rao, Anjana et al. (2016) LuxGLM: a probabilistic covariate model for quantification of DNA methylation modifications with complex experimental designs. Bioinformatics 32:i511-i519
Yue, Xiaojing; Trifari, Sara; Äijö, Tarmo et al. (2016) Control of Foxp3 stability through modulation of TET activity. J Exp Med 213:377-97

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