In 2009, my laboratory discovered the enzymatic activities of the three mammalian TET (Ten-Eleven-Translo- cation) proteins, TET1, TET2 and TET3. Since then, work from our own and other groups has implicated TET proteins in regulating gene expression, cell lineage specification, embryonic development, neuronal function and cancer. TET proteins are dioxygenases that oxidize 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmC) and further oxidized methylcytosines (oxi-mC). They have two biochemical functions: to generate oxi- mC and to facilitate DNA demethylation. However, the mechanisms by which TET proteins exert their diverse biological effects are much less understood. TET2 mutations are frequently observed in myeloid malignancies, but many other cancers are documented to have low 5hmC levels, implying profound loss of TET function even in the absence of TET coding region muta- tions. Because TET loss-of-function is associated with increased DNA methylation, the tumor suppressive role of TET proteins has been assumed to involve their ability to maintain DNA in a demethylated state. However, in powerful, inducible mouse models developed in our laboratory, we have shown that acute deletion of both Tet2 and Tet3 rapidly induces an aggressive, transmissible myeloid leukemia within 4 weeks and with 100% penetrance. Using this system, we have found that while the average level of DNA methylation increases across expressed genes in early hematopoietic stem/precursor cells as expected, there is little or no correla- tion of increased or decreased DNA methylation with up- or down-regulation of gene expression or with onco- genesis. Instead, we observe a strong correlation of oncogenic transformation with increased phospho-H2AX and impaired DNA damage repair. Here we propose to extend these studies to address the mechanisms involved. In this project we will use mouse models as well as in vitro systems to analyze the mechanisms of oncogenesis induced by TET loss-of-function. We will examine the role of TET catalytic activity, and compare the conse- quences of loss-of-function of TET proteins versus DNA methyltransferases (DNMTs). We will examine the kinetic relation between loss of oxi-mC in expanding cells and the development of replication stress, genome instability and chromosomal aberrations. As feasible, we will perform RNAi/CRISPR screens to identify important players that regulate cell expansion induced by TET loss-of-function. We will extend our findings to human cancers with high and low 5hmC. Our studies have the potential to change current paradigms and suggest new therapeutic approaches, by defining the mechanisms by which TET function is linked to genome stability.

Public Health Relevance

In addition to the four major bases in the DNA alphabet ? A, C, G and T ? there is also a ?fifth? base known as 5-methylcytosine that has a disproportionately crucial role. This base is produced by attaching a methyl group to the ?5? position of the major base C (cytosine). We recently identified a new class of proteins known as TET proteins that convert 5-methyl-cytosine to 5- hydroxymethylcytosine and other oxidized methylcytosines. Deficiencies of cytosine methylation and TET protein function are associated with developmental abnormalities, genetic diseases and cancer. In particular, we have shown that profound loss of TET function is strongly linked to cancer, both in humans and in mouse model systems. In this proposal we will investigate the mechanisms involved.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Unknown (R35)
Project #
1R35CA210043-01
Application #
9186697
Study Section
Special Emphasis Panel (ZCA1-GRB-I (M2))
Program Officer
Okano, Paul
Project Start
2016-09-01
Project End
2023-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
1
Fiscal Year
2016
Total Cost
$864,061
Indirect Cost
$384,027
Name
La Jolla Institute
Department
Type
DUNS #
603880287
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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
Khoueiry, Rita; Sohni, Abhishek; Thienpont, Bernard et al. (2017) Lineage-specific functions of TET1 in the postimplantation mouse embryo. Nat Genet 49:1061-1072
Scott-Browne, James P; Lio, Chan-Wang J; Rao, Anjana (2017) TET proteins in natural and induced differentiation. Curr Opin Genet Dev 46:202-208
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:
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