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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Oncogene. 2015 Feb 2;34(43):5436–5446. doi: 10.1038/onc.2014.472

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis

Staci L Haney 1,#, Ryan A Hlady 2,+,#, Jana Opavska 2, David Klinkebiel 3, Samuel J Pirruccello 4, Samikshan Dutta 3, Kaustubh Datta 3, Melanie A Simpson 5, Lizhao Wu 6, Rene Opavsky 1,2,7,#
PMCID: PMC4533871  NIHMSID: NIHMS711378  PMID: 25639876

Abstract

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified up-regulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3aΔ/Δ mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild-type and catalytically inactive Dnmt3a in Dnmt3aΔ/Δ lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b up-regulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3aΔ/Δ mice, demonstrating that up-regulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

Keywords: Mouse models, T-cell lymphoma, DNA methylation, Epigenetics

INTRODUCTION

Methylation of CpG dinucleotides in DNA is an essential epigenetic modification involved in X-chromosome inactivation, genomic imprinting, and tissue specific gene regulation.1 Three DNA methyltransferases (Dnmts) catalyze the addition of a methyl group to cytosine in mammalian cells: Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is primarily responsible for maintenance of methylation patterns during cellular divisions through its ability to read and transfer methylation groups to the newly synthesized DNA strand during replication.2 Consistently with the importance of maintaining epigenetic integrity in dividing cells, homozygous deletion of Dnmt1 is lethal at early stages of embryogenesis.3 Dnmt3a and Dnmt3b function primarily as de novo enzymes.4 Dnmt3b is responsible for early de novo methylation and repression of germ line genes5 and its inactivation is embryonically lethal. Dnmt3a is dispensable for embryogenesis but Dnmt3a knockout mice die shortly after birth due to multiple organ failure.4 Emerging evidence suggests that all three enzymes may play a role in maintenance and de novo activity in a locus specific manner1 but their ability to affect promoter methylation in normal and pathological settings is still poorly understood.

Dnmt3a and Dnmt3b share several sequence and structural similarities, including a conserved C-terminal domain, which mediates addition of methyl groups to DNA and the N-terminal regulatory domain that mediate interactions with DNA and other proteins.1 Methylation at CpG dinucleotides found in the promoter and other regulatory regions is often associated with transcriptional silencing.1 In addition to their methyltransferase activities, Dnmt3a and Dnmt3b can repress transcription in a methylation-independent manner. Critical to this process is their interaction with histone deacetylases (HDACs) and other repressor proteins via their ATRX-like domain.6 For instance, Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and Brg1 to silence metallothionein-I transcription in mouse lymphosarcoma cells.7 However, how methylation-independent repressor activity affects physiological processes remains elusive.

Genome-wide deregulation of the DNA methylation landscape, including locus-specific hyper- and global hypo-methylation, is a consistently observed phenomenon in human tumors. This deregulation, in particular hypomethylation, likely comes from genetic alterations of DNMTs found in cancer. While mutations in DNMTs were identified in a variety of human tumors they are most often found in hematologic malignancies. For example, DNMT3A is one of the most frequently mutated genes in myeloid and T-cell malignancies with the frequency ranging from 8% cases of myelodysplastic syndrome (MDS) to 33% of angioimmunoblastic T-cell lymphoma.8, 9, 10, 11 In T-cell malignancies, approximately 2/3 of mutations are missence, with the remainder being frame shifts, nonsense mutations, and deletions. 9, 11, 12 The majority of mutations cluster in the catalytic domain, suggesting that the change in methylase activity is important for tumor development. The substitution of arginine for histidine in the catalytic domain (R882H mutation) accounts for ~60% of the mutations in acute myeloid leukemia (AML). In addition to being hypomorphic, this mutant is believed to function as a dominant negative protein.13 The effects of aberrations found outside the catalytic domain, and those common to T-cell malignancies, are not known. In contrast to DNMT3A, DNMT3B or DNMT1 are rarely mutated in hematologic malignancies. Why in hematologic diseases genetic alterations are present in DNMT3A but not in other DNMTs is unclear. Like mutations in DNMT3A, increased activity of the proto-oncogene MYC is frequently observed in human T-cell malignancies, either through mutations in oncogenes, such as NOTCH1 for which MYC is a transcriptional target, or through genetic alterations of the MYC locus itself.14 For example, a subset of peripheral T-cell lymphomas (TCLs) has a frequent gain of the MYC locus (8q24) with subsequent overexpression, suggesting that MYC plays a role in the pathogenesis of the disease.15

The sensitivity of T-cells to MYC-induced transformation was previously demonstrated using a bitransgenic EμSRα-tTA;Teto-MYC mouse model in which tetracycline transcriptional transactivator (tTA) drives MYC expression resulting in the development of immature TCLs.16 Using a model of MYC-induced T-cell lymphomagensis (MTCL), we recently demonstrated that conditional inactivation of Dnmt1 compromised normal and malignant hematopoiesis and delayed MYC-induced T-cell lymphomagenesis.17 In contrast, Dnmt3b functions as a tumor suppressor (TS) as its loss does not affect normal T-cell development but accelerates MTCL.18

Utilizing a model of MTCL, we show that loss of Dnmt3a extends the survival of mice due to a decrease in cellular proliferation with no effect on the disease spectrum. Using genome-wide approaches we observed up-regulation of TS genes, including Dnmt3b, E2f2, and Pten, whose expression is elevated in Dnmt3a-deficient lymphomas without apparent changes in DNA methylation in their promoters or gene bodies. We further show that catalytically inactive Dnmt3a inhibits Dnmt3b in vitro. Finally, genetic inactivation of Dnmt3b accelerated MTCL, suggesting that delayed lymphomagenesis is at least in part mediated by Dnmt3b. Altogether; our data provide evidence for an unexpected oncogenic function of Dnmt3a in MTCL, through methylation independent repressor activity critical for the proliferation of tumor cells.

RESULTS

Oncogenic role of Dnmt3a in MYC-induced T-cell lymphomagenesis (MTCL)

To evaluate the role of Dnmt3a in MTCL we generated EμSRα-tTA;Teto-MYC; Rosa26LOXPEGFP/EGFP;Dnmt3aF/F (designated MYC;Dnmt3aF/For control mice), EμSRα-tTA;Teto-MYC; Teto-Cre;Rosa26LOXPEGFP/EGFP; Dnmt3a+/F (designated MYC;Dnmt3a+/Δ or heterozygous mice) and EμSRα-tTA;Teto-MYC;Teto-Cre;Rosa26LOXPEGFP/EGFP; Dnmt3aF/F (designated MYC;Dnmt3aΔ/Δ or Dnmt3a-deficient; Figure 1a) mice and measured survival. Although loss of one allele of Dnmt3a had no effect on MTCL, we found that the biallelic inactivation of Dnmt3a significantly extended the survival of mice relative to controls (Figure 1b). Analysis of protein and DNA confirmed the complete ablation of Dnmt3a in tumors arising in MYC;Dnmt3aΔ/Δ mice (Figures 1c and d). Loss of Dnmt3a had no effect on MYC expression (Supplementary Figure S1), suggesting that the delayed MTCL in Dnmt3a-deficient mice was not caused by down-regulation of transgenic MYC. Tumor burden in various hematopoietic organs was similar between control and Dnmt3a-deficient mice, indicating that loss of Dnmt3a did not affect mouse survival in a tissue-specific manner (Supplementary Figure S2). Like control lymphomas, tumors from MYC;Dnmt3aΔ/Δ were either CD4+/CD8+ or CD4+, with no measurable differences in expression of other T-cell (CD3, TCRβ, TCRγδ), B-cell (B220, CD19), myeloid (CD11b, Gr-1) or erythroid (TER119) markers (Figure 1e and data not shown). Altogether, this suggests that loss of Dnmt3a did not affect the disease spectrum in this MTCL model. Thus, contrary to our expectations, these data show that loss of Dnmt3a suppresses MTCL, suggesting an oncogenic function for Dnmt3a in T-cells.

Figure 1.

Figure 1

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Ablation of Dnmt3a delays T-cell lymphomagenesis. (a) Genetic setting used to conditionally delete Dnmt3a. The tetracycline activator protein (tTA) is expressed and promotes simultaneous expression of the Teto-MYC and Teto-Cre transgenes. Expression of Cre results in excision of the stop cassette located upstream of the Rosa26LOXPEGFP reporter locus and deletion of Dnmt3a within the same subpopulation of cells. Thus, inclusion of the EGFP transgene allows for monitoring cells expressing tTA, Cre and MYC, as well as to identify cells deleted for Dnmt3a. In the pre-tumor stage (from birth to approximately day 21) the EμSRα-tTA transgene is expressed in 30-50% of cell in all hematopoietic lineages, including hematopoietic stem cells. During tumorigenesis, MYC expressing cells expand rapidly, resulting in EGFP-positive tumors. (b) Kaplan-Meier survival curve for control (MYC;Dnmt3aF/F, blue), Dnmt3a-deficient (MYC;Dnmt3aΔ/Δ, red) and Dnmt3a heterozygous (MYC;Dnmt3a+/Δ, green) mice. Number of mice (n) and Median survival (MS) is shown. Comparison of MYC;Dnmt3aF/F to MYC;Dnmt3aΔ/Δ was statistically significant (P=0.0002). (c) Immunoblot showing Dnmt3a expression in normal thymocytes (N), control tumors (MYC;Dnmt3aF/F), and Dnmt3a-deficient tumors (MYC;Dnmt3aΔ/Δ). γ-Tubulin is shown as a loading control. (d) PCR based deletion efficiency of Dnmt3a in MYC;Dnmt3aΔ/Δ tumors. Dnmt3aF/Δ served as a control. F and Δ indicate floxed and knockout alleles, respectively. (e) FACS analysis of immunophenotypes is plotted to show percentage of CD4+ and CD4+CD8+ malignanies in MYC;Dnmt3aF/F (F/F) and MYC;Dnmt3aΔ/Δ (Δ/Δ) mice.

Dnmt3a inactivation decreases cellular proliferation during disease progression

We next sought to determine the nature of biological processes affecting MTCL in the absence of Dnmt3a. We first looked at T-cell development in 21 day old EμSRα-tTA;Teto-MYC;Teto-Cre;ROSA26EGFP/EGFPDnmt3a+/+ (MYC;Dnmt3a+/+) and MYC;Dnmt3aΔ/Δ mice. Although Dnmt3a was efficiently deleted in EGFP+ cells isolated from the thymi of MYC;Dnmt3aΔ/Δ mice (Figure 2a), no substantial differences were found in size or cellularity of thymi and spleens when compared to MYC;Dnmt3a+/+ in 21day old mice (data not shown). T-cell development evaluated by expression of CD4, CD8, CD25 and CD44 markers and the percentage of cells expressing EGFP was not affected by loss of Dnmt3a at 21 days (Figure 2b, Supplementary Figures S3 and S4). These data suggest that Dnmt3a is dispensable for T-cell development, consistent with our previous report.19 Similarly, no differences in the levels of apoptosis were observed between MYC;Dnmt3a+/+ and MYC;Dnmt3aΔ/Δ mice at any stage of tumor development (Figure 2c). In contrast, whereas BrdU incorporation was similar at early stages of tumor development (21 days), a substantial decrease in cells incorporating BrdU was observed at later stages of tumor development (35 days and terminally ill mice; Figure 2d and e, Supplementary Figures S5 and S6). Together, these results imply that the extended survival of MYC;Dnmt3aΔ/Δ mice is caused by decreased proliferation during disease progression.

Figure 2.

Figure 2

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Dnmt3a inactivation decreases cellular proliferation during disease progression. (a) PCR based deletion efficiency of Dnmt3a in EGFP+ sorted thymoctyes isolated from 21 day old MYC;Dnmt3aΔ/Δ mice. Dnmt3aF/Δ served as a control. F and Δ indicate floxed and knockout alleles, respectively. (b) FACS analysis of T-cell development in thymocytes isolated from 21 day old MYC;Dnmt3a+/+ (blue) and MYC;Dnmt3aΔ/Δ (red) mice. Number of mice (n) in each cohort is shown. Quantification of obtained results is shown as an average value with error bars representing ± standard error of the mean (SEM). (c) Analysis of apoptosis by Annexin V staining in cells isolated from the thymi of 21 day old, 35 day old, and final tumors in MYC;Dnmt3a+/+ (blue) and MYC;Dnmt3aΔ/Δ (red) mice. Error bars denote ± SEM. Number of mice used for each group is shown inside the bars. (d) Representative FACS diagrams showing BrdU labeling in MYC;Dnmt3a+/+ (blue) and MYC;Dnmt3aΔ/Δ (red) final thymic tumors. The percentage of cells staining positive is shown. (e) Quantification of BrdU-positive cells isolated from the thymi of 21 day old, 35 day old, and final tumors in MYC;Dnmt3a+/+ (blue) and MYC;Dnmt3aΔ/Δ (red) mice. Quantification of results for each timepoint is shown as an average value with error bars representing ± SEM. Number of mice used for each comparison is shown inside the bar. P<0.05 is denoted by a (*).

Dnmt3a ablation results in genome-wide methylation changes in lymphomas

To investigate the locus-specific effects of Dnmt3a on DNA methylation we used methyl-sensitive cut counting (MSCC) to profile the methylation patterns of MYC;Dnmt3aF/F and MYC;Dnmt3aΔ/Δ lymphomas along with normal Dnmt3a+/+ and Dnmt3aΔ/Δ thymocytes as previously described.17, 18, 19, 20 In this next-generation sequencing-based method, the methylation - evaluated based on the number of sequence tags (termed counts) - inversely correlates with the degree of methylation at HpaII and HpyCh4IV sites.20 A total of 24,236 promoters in the mouse genome have at least two HpaII and/or HpyCh4IV restriction sites. To rigorously assess the methylation status of promoters, we considered a change in methylation to be significant only if it occurred in two or more independent restriction sites in promoter areas from −1,500 to +500 base pairs relative to the transcription start site (≥ 2-fold, false discovery rate (FDR)<0.05). We first compared promoter-specific methylation between MYC;Dnmt3aF/F and MYC;Dnmt3aΔ/Δ lymphomas. This analysis revealed that 370 gene promoters were hypomethylated in MYC;Dnmt3aΔ/Δ lymphomas, suggesting that these promoters may represent potential targets of Dnmt3a-specific methylase activity (Figure 3a and Supplementary File S1). In contrast, the promoters of 64 genes were hypermethylated in MYC;Dnmt3aΔ/Δ lymphomas, likely as an indirect effect of Dnmt3a loss (Figure 3a).

Figure 3.

Figure 3

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Dnmt3a ablation results in genome-wide methylation changes in MYC;Dnmt3aΔ/Δ lymphomas. (a) Analysis of MSCC data showing 370 hypomethylated and 64 hypermethylated promoters in MYC;Dnmt3aΔ/Δ tumors relative to MYC;Dnmt3aF/F tumors. Proposed de novo (17 genes) and maintenance (353 genes) targets are labeled. Methylation status of normal thymocytes (N) is also shown. Promoter is defined as +500 to −1,500 relative to the transcription start site. Differentially methylated sites had a fold change of 2 or greater and an FDR < 0.05 at two independent CpG sites. Number of samples per group (n) is shown. A color bar depicting fold change is shown with blue representing a high degree of methylation and yellow representing lower levels. (b) COBRA analysis of the AK046742 and Leng1 promoters in normal thymocytes (N), MYC;Dnmt3aF/F and MYC;Dnmt3aΔ/Δ tumors. PCR fragments were digested with the restriction enzyme BstUI. Undigested {U} and digested {D} fragments correspond to un-methylated and methylated DNA, respectively. C indicates a fully methylated control. Position of primers relative to the transcription start site for each gene is shown in brackets to the left.

Next, we analyzed the methylation status of these promoters in normal thymocytes and Dnmt3a-deficient thymocytes. Out of the 370 promoters hypomethylated in MYC;Dnmt3aΔ/Δ lymphomas, 353 were also hypomethylated when compared to either normal or Dnmt3aΔ/Δ thymocytes, implying that Dnmt3a is dispensable for their de novo methylation during normal development, but may play a role in their maintenance methylation during tumorigenesis (Figure 3a and Supplementary Figure S7). In contrast, 17 promoters were specifically hypermethylated in MYC;Dnmt3aF/F tumors, indicating that they may represent potential targets of Dnmt3a cancer-specific de novo methylation (Figure 3a and Supplementary Figure S7). Finally, promoters of ten genes were hypomethylated in normal Dnmt3a-deficient thymocytes, suggesting that their methylation is already decreased during normal development and thus their hypomethylation in MYC;Dnmt3aΔ/Δ lymphomas is not tumor-specific (Supplementary Figure S7). Methylation readouts from MSCC analysis were confirmed for selected promoters using locus-specific combined bisulfite restriction analysis (COBRA). For example, the predicted gene AK046742 appeared to be a target of Dnmt3a’s cancer-specific de novo activity, as its promoter region was not methylated in normal thymocytes and MYC;Dnmt3aΔ/Δ tumors but was methylated in MYC;Dnmt3aF/F tumors (Figure 3b). Conversely, Leng1 may be a target of Dnmt3a maintenance activity as the area near its promoter was hypermethylated in normal thymocytes and MYC;Dnmt3aF/F lymphomas but not in MYC;Dnmt3aΔ/Δ lymphomas (Figure 3b). These data indicate that the contribution of Dnmt3a to the tumor-specific methylation patterns consists of both de novo and maintenance activities.

It is worth noting that the magnitude of Dnmt3a effects on genome-wide methylation may be larger, as analysis of promoters using single HpaII or HpyCh4IV sites showed 1,854 hypomethylated promoters in MYC;Dnmt3aΔ/Δ lymphomas (Supplementary Figure S8). Thus, a higher resolution genome-wide methylation profiling studies - such as whole-genome bisulfite sequencing - may better address this point in future studies.

DNA Methylation-independent up-regulation of tumor suppressor genes in Dnmt3a-deficient lymphomas

To further understand the molecular basis for the extended survival of MYC;Dnmt3aΔ/Δ mice, we compared microarray-based gene expression profiles of MYC;Dnmt3aΔ/Δ lymphomas to those of normal thymocytes and MYC;Dnmt3aF/F lymphomas. We identified 2,246 genes whose expression levels were significantly different (1.5-fold; P<0.05) between MYC;Dnmt3aF/F and MYC;Dnmt3aΔ/Δ lymphomas (Figure 4a, Supplementary File S2). Loss of Dnmt3a resulted in the transcriptional up-regulation of 1,421 genes, which is consistent with the function of Dnmt3a as a repressor protein. qRT-PCR confirmed that Cd79b, E2f2, and Pten were up-regulated in MYC;Dnmt3aΔ/Δ lymphomas (Figure 4b). Microarray analysis also identified 825 genes down-regulated in MYC;Dnmt3aΔ/Δ tumors (Figure 4a). Although we cannot rule out the possibility that Dnmt3a plays a role in transcriptional activation, these genes likely represent secondary changes resulting from deregulated transcription upon loss of Dnmt3a.

Figure 4.

Figure 4

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Loss of Dnmt3a leads to deregulated transcription. (a) A heat map derived from global transcription profiling by microarray displaying 1,421 over-expressed and 825 under-expressed genes in MYC;Dnmt3aΔ/Δ tumors relative to MYC;Dnmt3aF/F tumors (fold change ≥ 1.5 and P<0.05 by Bayesian t test). Expression in normal thymocytes (N) is also shown. A color bar is shown to reference fold change with up-regulation in red and down-regulation in green. Number of samples (n) is shown. (b) On the left, expression data obtained from microarray for three differentially expressed genes in MYC;Dnmt3aΔ/Δ (red) relative to MYC;Dnmt3aF/F (blue) tumors is shown. On the right, qRT-PCR data displaying the relative mRNA levels of the three genes is shown. Quantification of obtained results is shown as an average value with error bars representing ± SEM. Number of samples used for each comparison is shown inside the bars. P-values are shown with statistical significance denoted by a (*).

To gain further molecular insight into the pathogenesis of MYC;Dnmt3aΔ/Δ lymphomas, we performed Ingenuity Pathway Analysis (IPA) using the 1,421 genes that were significantly up-regulated relative to MYC;Dnmt3aF/F lymphomas. The top five disease groups associated with up-regulated genes were immunological disease, cancer, inflammatory disease, infectious disease, and hematological disease (Supplementary Figure S9). This search also identified a 17 gene signature under the disease network “Lymphomagenesis” whose up-regulation was predicted to suppress lymphomagenesis (Figure 5a). This signature consisted of Bcl2l11, Brca2, Dna2, Dnmt3b, E2f1, E2f2, Exo1, Irf1, Irf8, Nqo1, Prdm2, Pten, Recql4, Smurf2, Ssbp2, Tyk2, and Xrcc2. In contrast, analysis using genes down-regulated in MYC;Dnmt3aΔ/Δ lymphomas did not yield any significant change under the disease network “Lymphomagenesis”. Surprisingly, both promoter and gene body methylation was unaffected for all 17 genes (Figure 5b and Supplementary Figure S10), suggesting that the increased expression of these genes is independent of changes in DNA methylation. Furthermore, methylation of gene bodies (defined as +500 to the end of the gene) was largely unaffected by loss of Dnmt3a (Supplementary Figures S11 and S12). The 17 gene TS signature observed in Dnmt3a-deficient MTCLs may represent molecular events functionally contributing to the extended survival observed in MYC;Dnmt3aΔ/Δ mice.

Figure 5.

Figure 5

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DNA Methylation-independent up-regulation of tumor suppressor genes in Dnmt3a-deficient lymphomas. (a) The 17 gene signature “Lymphomagenesis” derived from Ingenuity Pathway Analysis (IPA) of 1,421 up-regulated genes in MYC;Dnmt3aΔ/Δ tumors. Upregulation of these 17 genes are predicted to suppress lymphomagenesis. Fold changes derived from microarray data, as well as Z-score and P-value generated by IPA are shown. (b) Bar graph showing quantification of promoter methylation for the 17 genes using average counts from MSCC data in MYC;Dnmt3aF/F (blue) and MYC;Dnmt3aΔ/Δ (red) tumors. Proposed Dnmt3a target gene, AK046742, is shown as a positive control. The promoter is defined as −1,500 to +500 relative to the transcription start site. Error bars denote ± SEM. P<0.05 is shown by a (*).

Dnmt3a represses Dnmt3b expression independently of its catalytic activity

Our global approach identified TS genes under the disease network “Lymphomagenesis” whose increased expression is associated with little-to-no changes in DNA methylation. Thus, their increased expression could either be in a direct response to loss of Dnmt3a repressor function or could be an indirect consequence of lymphomagenesis. We have recently reported that a close relative of Dnmt3a, Dnmt3b, is a TS gene in this MTCL model due to its ability to negatively regulate cellular proliferation during disease progression.18 Since our data indicate that a key biological process behind delayed MTCL is decreased proliferation and our global gene expression data showed up-regulation of Dnmt3b in MYC;Dnmt3aΔ/Δ tumors, we wondered whether Dnmt3a regulates Dnmt3b expression. To address this we first looked at the expression of Dnmt3b in MYC;Dnmt3aΔ/Δ lymphomas. This analysis revealed up-regulation of mRNA and protein levels of Dnmt3b, suggesting that Dnmt3a may repress Dnmt3b (Figure 6a and b). Re-introduction of wild-type Dnmt3a into cell lines derived from MYC;Dnmt3aΔ/Δ lymphomas resulted in moderate but significant decrease in Dnmt3b RNA and protein levels (Figure 6c, d, e, Supplementary Fig. S13). Since we did not detect differences in methylation levels of the Dnmt3b promoter or gene body, we asked whether Dnmt3a inhibition of Dnmt3b could be independent of its methylase activity. To test this directly we infected MYC;Dnmt3aΔ/Δ lymphoma cells with Dnmt3a in which two key amino acids in the catalytic domain were mutated to produce a catalytically dead Dnmt3a protein (Dnmt3aCD).21 Overexpression of Dnmt3aCD repressed Dnmt3b expression to a similar extent as wild-type Dnmt3a. Altogether, these data suggest that Dnmt3a represses Dnmt3b in a methylation-independent manner (Figure 6c, d, e).

Figure 6.

Figure 6

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Dnmt3a represses Dnmt3b expression independently of its catalytic activity. (a) qRT-PCR analysis of Dnmt3b transcript levels in control MYC;Dnmt3aF/F tumors (blue) and MYC;Dnmt3aΔ/Δ tumors (red). The number of samples used for each group is shown inside the bars. Error bars denote ± SEM. P<0.05 is shown by a (*). (b) Immunoblot showing Dnmt3b protein levels in MYC;Dnmt3aF/F tumors, MYC;Dnmt3aΔ/Δ tumors, and a Dnmt3b-deficient control (b). PCNA served as a loading control. (c) Immunoblot analysis showing Dnmt3b and Dnmt3a expression in three independent MYC;Dnmt3aΔ/Δ cell line (A, B, C) infected with empty vector (Vec), wild-type Dnmt3a (3a), or catalytically dead Dnmt3a (3aCD). Lane 4 shows a Dnmt3b-deficient control (NC). PCNA and γ-Tubulin are shown as loading controls. (d) Quantification of Dnmt3b protein levels in MYC;Dnmt3aΔ/Δ cell lines (A, B, C) for Figure 6c. Dnmt3b levels were normalized to PCNA. Relative protein levels were calculated by adjusting values for empty vector control (Vec) to one and using this as a baseline for all other comparisons. (e) qRT-PCR analysis of Dnmt3a (left) and Dnmt3b (right) transcript levels in cell lines derived from MYC;Dnmt3aΔ/Δ tumors infected with empty vector control (black), wild-type Dnmt3a (red), or catalytically dead Dnmt3a (yellow). Error bars represent ± SEM. P-values are shown with statistical significance denoted by a (*).

Loss of Dnmt3b accelerates lymphomagenesis in Dnmt3a deficient mice

We next hypothesized that up-regulation of Dnmt3b may be an important molecular event inhibiting MTCL in the absence of Dnmt3a. To test this directly, we generated and compared the survival of MYC;Dnmt3aΔ/Δ;Dnmt3bΔ/Δ (Double-knockout; DKO) mice to MYC;Dnmt3aF/F;Dnmt3bF/F mice (Figure 7a). In contrast to the prolonged survival seen in MYC;Dnmt3aΔ/Δ mice, DKO mice displayed indistinguishable survival compared to MYC;Dnmt3aF/F;Dnmt3bF/F control mice (Figure 7b). Both Dnmt3a and Dnmt3b were efficiently inactivated in DKO lymphomas (Figure 7c and d). Apoptosis, T-cell development, tumor burden, transgenic MYC expression, and tumor spectrum were similar between terminally sick control and DKO mice (Supplementary Figures S14-19), indicating that the additional loss of Dnmt3b did not impact these processes. Interestingly, proliferation of lymphoma cells, a process impaired in MYC;Dnmt3aΔ/Δ mice, was similar between terminally sick control and DKO mice (Figure 7e), which is consistent with the decreased survival of DKO mice relative to Dnmt3a-deficient mice. The distinct roles of Dnmt3a and Dnmt3b in MTCL are consistent with molecular changes observed in Dnmt3a- or Dnmt3b-deficient lymphomas. Out of 370 promoters hypomethylated in MYC;Dnmt3aΔ/Δ lymphomas, only 3% were hypomethylated in Dnmt3b-deficient lymphomas18, indicating that Dnmt3a and Dnmt3b have distinct targets in vivo (Figure 7f). Similarly, only 11% of genes up-regulated in Dnmt3a-deficient lymphoma were also overexpressed in Dnmt3b-deficient lymphomas (Figure 7f).18 Collectively, these data illustrate both cellular and molecular differences between Dnmt3a and Dnmt3b in MTCL.

Figure 7.

Figure 7

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Loss of Dnmt3b accelerates lymphomagenesis in Dnmt3a deficient mice. (a) Genetic setting used to delete conditional alleles of Dnmt3a and Dnmt3b. (b) Kaplan-Meier survival curve for MYC;Dnmt3aF/F;Dnmt3bF/F (blue), MYC;Dnmt3aΔ/Δ;MYC;Dnmt3bΔ/Δ (green) and MYC;Dnmt3aΔ/Δ (red, also shown in Figure 1b) mice. Median survival (MS) and number of mice (n) is shown. (c) Immunoblot showing Dnmt3a and Dnmt3b expression in control tumors (MYC;Dnmt3aF/F;Dnmt3bF/F), Dnmt3a;Dnmt3b-deficient tumors (MYC;Dnmt3aΔ/Δ;MYC;Dnmt3bΔ/Δ) and a Dnmt3b-deficient control (b). γ-Tubulin is shown as a loading control. (d) PCR based deletion efficiency of Dnmt3a and Dnmt3b in MYC;Dnmt3aΔ/Δ;Dnmt3bΔ/Δ tumors. Dnmt3aF/Δ and Dnmt3bF/Δ served as controls. F and Δ indicate floxed and knockout alleles, respectively. (e) Quantification of BrdU-positive cells isolated from final thymic tumors in MYC;Dnmt3aF/F;Dnmt3bF/F (blue) and MYC;Dnmt3aΔ/Δ;Dnmt3bΔ/Δ (green) mice. Error bars represent ± SEM. Number of mice used for each comparison is shown inside the bars. (f) Venn diagrams displaying the overlap between genes hypomethylated (left) and overexpressed (right) in both Dnmt3b- and Dnmt3a-deficient tumors. The number of gene identified by methylation analysis using MSCC (Fold change ≥ 2, FDR<0.05, Benjamini Hochberg) and expression analysis using microarray (Fold change ≥ 1.5, P<0.05, Bayesian t test), are shown.

DISCUSSION

Since the discovery of DNMT3A mutations in MDS and AML8, 10 a number of alterations in DNMT3A were found in other human hematologic malignancies, including T-cell leukemias and lymphomas.9, 11, 12 In both myeloid and lymphoid malignancies, mutations in DNMT3A primarily occur in the catalytic domain8-12, suggesting that inactivation of the methyltransferase activity contributes to transformation. A recent biochemical analysis showed that the most common mutation, DNMT3A R882H, decreases methylase activity by ~80% at least partially by disrupting the ability of Dnmt3a to homotetramerize.22 Overexpression of mouse R878H mutant (analogous to human R882H) in embryonic stem cells decreased the activity of wild-type Dnmt3a and Dnmt3b likely by functioning as a dominant-negative and forming complexes with wild-type Dnmt3a and Dnmt3b.13 Ectopic expression of DNMT3A R882H in hematopoietic stem cells followed by transplantation into lethally irradiated mice induced a disease resembling chronic myelogenous leukemia within one year, demonstrating direct functional involvement of mutated DNMT3A in the development of myeloid leukemia. Here we used a mouse genetic model that allowed us to evaluate the effects of Dnmt3a inactivation along with MYC overexpression in all hematopoietic lineages. Although overexpression of MYC typically results in the development of T-cell lymphomas, the development of AML in this model was reported in 13% of mice.16 We observed that Dnmt3a inactivation did not alter normal hematopoietic development, tumor spectrum or tumor type. Rather, Dnmt3a-deficiency inhibited MTCL due to inhibitory effects on proliferation during disease progression and in tumor cells.

Several interesting implications arise from these studies. For instance, Dnmt3a functions as both an oncogene and TS in the hematopoietic compartment. This finding is surprising in view of our recent study that long-term loss of Dnmt3a in hematopoietic cells results in B-cell transformation and the development of chronic lymphocytic leukemia (CLL) in mice.19 Thus, the Dnmt3a locus harbors both oncogenic (promotion of MTCL due to its pro-proliferative function) and TS (prevention of CLL development) functions in the hematopoietic compartment. What is the molecular basis for such different activities in similar cell types? We speculate that the differences may stem from two biological functions of Dnmt3a – methylation-independent and methylation-dependent repressor activities, respectively. This concept is supported in the MTCL model by IPA of microarray data which identified 17 genes under the category of “Lymphomagenesis” whose up-regulation in Dnmt3a-deficient lymphomas was predicted to suppress lymphomagenesis. Importantly, whereas all of these genes were overexpressed in MYC;Dnmt3aΔ/Δ tumors, we have not observed changes their promoters or gene body methylation. The lymphomagenesis signature consisted of genes whose TS functions were either reported in spontaneous B- and T-cell lymphomagenesis (Brca2, Dna2, Exo1,Prdm2, Smurf2, Ssbp2),23-28 p53-deficient or oncogene-provoked lymphomagenesis (Bcl2l11, E2f1, Xrcc2, Recql4, Tyk2),29-33 including MTCL (Pten, E2f2, Dnmt3b),34, 35, 18 or other aspects of lymphomagenesis (Nqo1, Irf1, Irf8).36-38 Whether up-regulation of any of these genes is involved in MTCL in the absence of Dnmt3a remains to be seen, however individual inactivation of E2f2 or Pten accelerated MTCL.34, 35 Furthermore, given that Xrcc2 deficiency accelerated lymphomagenesis induced by loss of p5331, and Brca2 deficiency results in the development of T-cell lymphomas23, other TS genes may be contributing to the extended survival of MYC;Dnmt3aΔ/Δ mice.

In contrast to MTCL, loss of Dnmt3a induces CLL and is associated with widespread promoter hypomethylation in this context.19 In view of these data, coupled with recently reported extensive promoter and gene body hypomethylation in human CLL,39 it seems that the basis for Dnmt3a TS function in CLL is its methyltransferase activity. Such a conclusion is further supported by the recent finding that the Tcl1 oncoprotein, whose overexpression induces CLL in mice inhibits Dnmt3a catalytic activity which likely contributes to disease development.40 An alternative possibility is that methylation-dependent and independent repressor activities of Dnmt3a play opposing roles in T- and B-cell transformation but T-cells are differentially sensitive due to cell-type specific differences. Dnmt3a was reported to function as a TS in lung cancer and as an oncogene in colorectal carcinoma,41, 42 further supporting that the role of Dnmt3a in tumorigenesis is highly complex and likely context specific. Here we show that Dnmt3a functions as an oncogene in MTCL, likely through the methylation-independent repression of TS genes.

Another conclusion from our studies is that Dnmt3a and its close relative Dnmt3b have distinct functions in MTCL. We have recently reported that loss of Dnmt3b, unlike Dnmt3a, accelerated MTCL.18 Others have shown that Dnmt3b haploinsufficiency promoted MYC-induced B-cell lymphomagenesis,43 clearly demonstrating a TS role for Dnmt3b. It was reported that loss of Dnmt3a results in exhaustion of stem cell self-renewal and defective differentiation.44 However, our data presented here and previously, point to a lack of differences of Dnmt3a or Dnmt3b deficiency on thymocyte development, likely due to different biological settings.18, 19 Thus, opposing roles of Dnmt3a and Dnmt3b on MTCL cannot be explained by changes in hematopoiesis. Rather, Dnmt3a and Dnmt3b seem to have contrasting effects on proliferation of T-cell lymphomas, with Dnmt3a promoting and Dnmt3b inhibiting proliferation of tumor cells. Molecularly, the scope of tumor specific changes in methylation and gene expression are larger for Dnmt3a compared to Dnmt3b. Consistent with the differential effects of these enzymes on MTCL, out of the 17 genes in the Dnmt3a-specific “Lymphomagenesis” signature, only one gene (Bcl2l11) is up-regulated in Dnmt3b-deficient MTCLs, further supporting the idea that at least some of these genes are responsible for the extended survival in MYC;Dnmt3aΔ/Δ mice. Whether these broader molecular effects of Dnmt3a in MTCL reflect different protein levels or qualitative differences in the functions of Dnmt3a and Dnmt3b remains to be seen.

Importantly, we present genetic evidence that the oncogenic function of Dnmt3a is at least in part, due to the negative regulation of the tumor suppressor Dnmt3b. The lack of methylation changes in the promoter and gene body, coupled with the ability of both wild-type and a catalytically dead mutant of Dnmt3a to repress Dnmt3b in vitro, suggest that methylation-independent repressor activity is important for its oncogenic functions. We show that repression is likely relevant in vivo, as genetic inactivation of Dnmt3b accelerates MTCL.

One critical question that remains to be answered is how the methylation independent activity of Dnmt3a represses Dnmt3b. Dnmt3a interacts with a number of repressor proteins, including HDAC1, Rb, and Daxx. 6, 45, 46 Thus, recruitment of Dnmt3a to the Dnmt3b promoter could bring repressors that inhibit Dnmt3b transcription. Direct binding of Dnmt3a to sequences in the Dnmt3b promoter seems unlikely given that no clear Dnmt3a binding site has been identified, although weak consensus sequences were reported. 47 Instead, interaction of Dnmt3a with transcription factors and subsequent recruitment to the Dnmt3b promoter seems more likely. Indeed, it was previously reported that the transcriptional repressor RP58 targeted Dnmt3a to a synthetic promoter to silence gene expression in a methylation independent manner.6 Dnmt3a could also interfere with the ability of transcription factors to activate transcription. For example, p53-mediated transactivation of the CDKN1A gene was suppressed by direct Dnmt3a interaction with p53 without changes in DNA methylation.48 Which protein mediates the potential recruitment of Dnmt3a to the Dnmt3b promoter is difficult to predict, given that Dnmt3a can interact with at least 68 transcription factors47, including c-Myc, Ets1, Gata1, Creb, and NF-KappaB, all of which have predicted binding sites in the Dnmt3b promoter region. Thus, future studies need to address this point.

It is also possible that rather than interacting with transcription factors, Dnmt3a interacts with histone modifying enzymes to induce repressive histone modifications. For examples, the N-terminal of Dnmt3a interacts with the histone methyltransferase SETDB1 to form a complex which binds at a synthetic promoter region and methylation of H3-K9 histones, but not of DNA, results in promoter inactivation.49

Finally, Dnmt3a was shown to directly interact with chromatin-remodeling factor Brg1, a subunit of the SWI/SNF complex that plays a role in both activation and repression of gene transcription.7 Thus, this interaction could induce nucleosomal rearrangement in the Dnmt3b promoter that would inhibit gene transcription in a DNA methylation independent manner.

Whatever the precise mechanism of Dnmt3a-mediated repression is, the unexpected role of Dnmt3a in MTCL raises the possibility that some of the mutations in DNMT3A located outside of the methyltransferase domain may enhance biological processes contributing to methylation-independent repression. To our knowledge, this is the first report highlighting the distinct but interconnected roles of Dnmt3a and Dnmt3b in cancer through methylation-independent repressor activity of Dnmt3a.

MATERIALS AND METHODS

Mouse studies

EμSRα-tTA;Teto-MYC and Dnmt3aF/F mice were obtained from D.W. Felsher (Stanford University) and R. Jaenisch (Whitehead Institute), respectively. ROSA26EGFP and Teto-Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All experiments were performed using mice of FVB/NJ background. Genotypes were confirmed by PCR using genomic DNA isolated from mouse tails. For analysis of T-cell development (including cell surface marker analysis, EGFP expression, and weights and cellularity of thymus, lymph node, and spleen) 21 day old mice from the EμSRα-tTA;Teto-MYC;Teto-Cre;Rosa26LOXPEGFP/EGFP;Dnmt3aF/F (MYC;Dnmt3aΔ/Δ), and EμSRα-tTA;Teto-MYC;Teto-Cre;ROSA26EGFP/EGFPDnmt3a+/+ (MYC;Dnmt3a+/+) cohorts were used. FACS-sorted EGFP+ thymocytes isolated from 21-day-old EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP/EGFP (Dnmt3a+/+) and EμSRα-tTA;Teto-Cre; Rosa26LOXPEGFP/EGFP;Dnmt3aF/F mice (Dnmt3aΔ/Δ) were used as controls for MSCC analysis. For tumor studies, the survival of EμSRα-tTA;Teto-MYC; Rosa26LOXPEGFP/EGFP;Dnmt3aF/F mice (MYC;Dnmt3aF/F) was compared to MYC;Dnmt3aΔ/Δ or EμSRα-tTA;Teto-MYC;Teto-Cre;Rosa26LOXPEGFP/EGFP;Dnmt3aF/+ (MYC;Dnmt3aΔ/+) mice and survival of EμSRα-tTA;Teto-MYC;Rosa26LOXPEGFP/EGFP; Dnmt3aF/F;Dnmt3bF/F (MYC; Dnmt3aF/F;Dnmt3bF/F), was compared to that of EμSRα-tTA;Teto-MYC;Teto-Cre;Rosa26LOXPEGFP/EGFP;Dnmt3aF/F;Dnmt3bF/F (MYC; Dnmt3aΔ/Δ; Dnmt3bΔ/Δ). Full genotypes with abbreviations and genetic crosses to produce mice are listed in Supplementary Tables S1 and S2. Differences in survival were calculated using the Kaplan-Meier method and the log-rank test for survival distributions. A two-sided Student’s t test was used to analyze differences in tumor burden, EGFP percentage, and cell surface marker expression.

Methyl-sensitive cut counting (MSCC) and data analysis

MSCC library preparation, data collection, and data analysis were performed as previously described.17, 18, 19, 20 The method results in an output of sequencing tags, or counts, which inversely correlate with the methylation status of a particular CpG. The R programming language and bioconductor package “edgeR” was used for statistical analysis of count data50, 51. A false discovery rate (FDR) was estimated using the Benjamini Hochberg method. To carefully assess the methylation status of promoters, we considered a change in methylation to be significant only if it occurred in two or more independent HpaII and/or HpyCh4IV restriction sites, with a fold change ≥ 2, at an FDR of < 0.05. The promoter was defined as −1500 to +500 base pairs relative to the transcription start site. Results were confirmed using COBRA as previously described.17, 18

Affymetrix microarray analyses

Microarray was performed at the UNMC Microarray core facility as previously described.18 Statistical analysis was done using Cyber-T software.52 Genes differentially expressed (P<0.05 and fold change >1.5) in MYC;Dnmt3aΔ/Δ tumors relative to MYC;Dnmt3aF/F tumors were analyzed using Ingenuity Pathway Analysis (Qiagen, Valencia, CA, USA) to identify common pathways and disease associations. Microarray data was deposited in NCBI’s Gene Expression Omnibus (accession no. GSE59338).

Generation of retroviruses and infection of cell lines

Retroviral vectors were created by subcloning the coding sequences of wild-type Dnmt3a and catalytically dead Dnmt3a (Dnmt3aCD) into the MSCV-IRES-RFP vector.21 Dnmt3aP705V/C706D has two amino acid substitutions in the catalytic domain which eliminate methylase activity of the enzyme.21 Constructs were verified by sequencing. Tumor cells isolated from MYC;Dnmt3aΔ/Δ mice were cultured in vitro in RPMI 1640 medium supplemented with 10% FBS and 0.025 mM 2-mercaptoethanol. Retroviral infection of cells was performed as previously described.18 Cells were infected using one of three retroviral vectors (MSCV-IRES-RFP, MSCV-IRES-Dnmt3a-RFP, or MSCV-IRES-Dnmt3aCD-RFP) and RFP+ cells were sorted 72 hours after infection for RNA isolation and qRT-PCR analysis.

Supplementary Material

Complete Supplemental Figures
Supplementary File 1
Supplementary File 2
Supplementary File 3
Supplementary Material
Supplementary Table 1
Supplementary Table 2

Footnotes

The authors disclose no conflict of interest.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Associated Data

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Supplementary Materials

Complete Supplemental Figures
NIHMS711378-supplement-Complete_Supplemental_Figures.pdf (2.3MB, pdf)
Supplementary File 1
NIHMS711378-supplement-Supplementary_File_1.xlsx (62.2KB, xlsx)
Supplementary File 2
NIHMS711378-supplement-Supplementary_File_2.xlsx (190.3KB, xlsx)
Supplementary File 3
NIHMS711378-supplement-Supplementary_File_3.xlsx (11KB, xlsx)
Supplementary Material
NIHMS711378-supplement-Supplementary_Material.docx (12.6KB, docx)
Supplementary Table 1
NIHMS711378-supplement-Table_1.pdf (48.9KB, pdf)
Supplementary Table 2
NIHMS711378-supplement-Table_2.pdf (90.7KB, pdf)

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