Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
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Research Article Hematology

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

Abstract

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

Authors

Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky

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Figure 8

Upregulation of Ment contributes to mouse and human lymphomagenesis.

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Upregulation of Ment contributes to mouse and human lymphomagenesis. (A)...
(A) Growth curves of MYC;Dnmt3b–/– cell lines infected with retroviruses encoding either control shRNA or independent shRNAs against Ment, and their corresponding cell cycle profiles 36 hours after plating. (B) Percent EGFP+ cells at different time points during in vitro growth of 1 control and 4 Ment-infected cell lines. Western blot of control and Ment-infected MYC;Dnmt3bfl/fl cells using anti-MYC antibodies is also shown. (C) siRNA-mediated knockdown of human MENT in JURKAT cells. Cells transfected with control siRNA or with SmartPool siRNA against MENT were counted 36 hours later. Data from 3 experiments were normalized to control; error bars represent ± SEM. P = 0.02, Student’s t test. (D) Expression of MENT, determined by qRT-PCR analysis of 42 human primary lymphomas present in TissueScan Lymphoma panel I (Origene). Data from 2 independent experiments are presented as fold difference relative to the average of 6 control samples. Red bars represent lymphomas with statistically significant fold increases in MENT expression. Below, methylation status of MENT locus I in 1 control and 1 lymphoma sample (corresponding to the bars marked with asterisks above), as determined by bisulfite sequencing. (E) Relative MENT transcript levels, as determined by qRT-PCR, in human breast, kidney, liver, and ovary tumors from the Origene TissueScan starter kit. Green bars represent control samples within each tissue. (A–E) Error bars represent ± SEM. *P < 0.05, Student’s t test.