Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

Abstract

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.

Keywords: DNA methylation; TET2; enhancer; leukemia.

© 2015 Rasmussen et al.; Published by Cold Spring Harbor Laboratory Press.

Figures

Figure 1.

Loss of Tet2 and AE expression collaborate to induce AML. (A) Serial replating assay in methylcellulose-containing medium of Tet2fl/fl or Tet2−/− Kit-enriched HSPCs transduced with either EV- or AE-expressing retrovirus. Bars represent mean value (n = 3), and error bars indicate SD. (*) P < 0.025 (two-way ANOVA); (n.s) not significant. (B) Kaplan-Meier plot showing overall recipient mouse survival upon primary (1°) or secondary (2°) transplantation. (Left) Lethally irradiated (900 Rad) recipient SJL mice transplanted with Tet2fl/fl (n = 13) or Tet2−/− (n = 12) Kit-enriched HSPCs transduced with AE-expressing retrovirus. (Right) Sublethally irradiated (650 Rad) recipient mice (n = 5) transplanted with 1 × 106 splenocytes isolated from moribund leukemic Tet2−/−;AE mice (LeuA). (****) P-value < 0.0001 (Wilcoxon test). LeuA and LeuB mark the origins of two Tet2−/−;AE leukemias from independent mice used for secondary transplantation experiments and gene expression analysis. (C) Peripheral blood parameters of wild-type (WT) mice (n = 4) or moribund mice transplanted with Tet2−/−;AE HSPCs (n = 7). (*) P < 0.01 (Student's t-test). (D) Splenomegaly observed in moribund mice transplanted with Tet2−/−;AE HSPCs, as indicated by spleen versus body weight ratio (n = 4) (left panel) or visual inspection (n = 3) (right panel). (***) P < 0.0001 (Student's t-test). (E) Representative FACS analysis plots of peripheral blood from recipient mice showing the CD45.2-positive Tet2fl/fl;AE (top panel) or Tet2−/−;AE (bottom panel) cells as well as the CD45.1-positive helper cells (wild type). Early signs of disease include the appearance of CD45-negative and GFP-positive leukemic blasts in circulation. (F) Unsupervised hierarchical clustering of mouse wild-type GMPs and leukemic GMPs (L-GMPs; LeuA and LeuB) together with human AML samples from the MILE study (Haferlach et al. 2010) (left) and the Cancer Genome Atlas (TCGA) study (The Cancer Genome Atlas Research Network 2013) (right). For clarity, each karyotypic subgroup in the MILE study is limited to 10 patients showing the highest intrasample correlation. In addition, TCGA patients with co-occurring mutations in multiple genes involved in DNA methylation (TET1, DNMT3A, DNMT3B, IDH1, and IDH2) were excluded, and only patients with TET2 mutations and/or t(8;21) translocations as well as an APL control group are shown. Samples with TET2 mutation are reported with red stars. See Supplemental Figure S1 for additional clustering analysis.

Figure 2.

Disruption of Tet2 in preleukemic AE cell cultures leads to gene expression changes similar to those found in human AML with TET2 mutations. (A) Overview of experimental setup (top) and quantification of global 5hmC levels by dot blotting (bottom). Genomic DNA isolated from biological duplicate cultures (Rep1 and Rep2) of Tet2fl/fl;AE;CreER cells treated with EtOH (control) or 4-OHT (induction of Tet2 disruption) were assayed. To show specificity, genomic DNA from wild-type or DNMT triple-knockout embryonic stem (ES) cells (Tsumura et al. 2006) as well as synthetic oligonucleotides were included. (B) Representative FACS plots showing populations of immature iGMP cells (GFP+CD3e−B220−Gr1−Mac1−CD16/32+) as well as mature Gr1+Mac1+ myeloid cells. Both Tet2fl/fl;AE (top panel) and Tet2−/−;AE (bottom panel) cultures show sign of granulocytic differentiation. (C) Accelerated in vitro proliferation of AE cells upon disruption of Tet2. Cumulative cell numbers or average doubling time (inset) of Tet2fl/fl;AE and Tet2−/−;AE cell cultures (n = 3). Cells were counted and plated at equal cell density every second day for a period of 20 d. Symbols show mean cell number, and error bars indicate SD. (****) P < 0.0001 (nonlinear regression). (D) Heat map of the top differentially expressed genes (false discovery rate [FDR] < 0.05, fold change >2) in sorted iGMP cells isolated from triplicate Tet2fl/fl;AE and Tet2−/−;AE cultures grown for two and 10 passages, respectively. Individual expression values were normalized, and the z-scores for each gene are presented. See Supplemental Table S2 for a full list of differentially expressed genes. (E) Tet2−/−;AE cells cluster closely with TET2 mutated AML patients, indicating that gene expression changes observed in iGMP cells upon Tet2 deletion are relevant to gene signatures of TET2 mutated AML patients. Differential gene expression-based hierarchical clustering of preleukemic Tet2fl/fl;AE and Tet2−/−;AE cells together with patients from TCGA (The Cancer Genome Atlas Research Network 2013) are shown. For clarity, patients with co-occurring mutations in multiple genes involved in DNA methylation (TET1, DNMT3A, DNMT3B, IDH1, and IDH2) were excluded, and only patients with TET2 mutations and/or t(8;21) translocations as well as an APL control group are shown. See also Supplemental Figure S2 for additional clustering analysis.

Figure 3.

5hmC is specifically lost at enhancers. (A) Heat map showing an overview of 5hmC-DIP-seq read density on all 8739 enhancers in duplicate cultures of Tet2fl/fl;AE and Tet2−/−;AE cells. The top panel shows 2999 enhancers enriched in 5hmC in wild-type and depleted of 5hmC upon Tet2 knockout (mean log2 fold change >0.5), whereas the bottom panel shows the remaining enhancers. Each row represents a 20-kb window centered on an enhancer and extends 10 kb upstream and 10 kb downstream. (B) Summarized 5hmC-DIP-seq read densities across all 8739 enhancers in duplicate cultures of Tet2fl/fl;AE and Tet2−/−;AE cells. See Supplemental Figure S3 for other genomic elements. (C) Representative UCSC tracks showing specific loss of a 5hmC peak in an enhancer in the Zfp128 locus. The top tracks show 5hmC-DIP-seq enrichment data from biological duplicate cultures of Tet2fl/fl;AE and Tet2−/−;AE, respectively. The bottom tracks represent histone ChIP-seq experiments performed on Tet2fl/fl;AE and Tet2−/−;AE cells showing enrichment of H3K27ac, H3K4me1, and H3K4me3, respectively. (D) 5hmC-DIP followed by qPCR in independent biological triplicate samples. Primers directed against the Zfp128 enhancer as well as an unchanging downstream peak are shown. Bars represent mean enrichment over input, and error bars indicate SD. (*) P-value < 0.05 (Student's t-test). See also Supplemental Figure S3 for validation of additional loci.

Figure 4.

eRRBS reveals preferential DNA hypermethylation in enhancer elements. (A) DNA methylation scatter plot showing methylation levels of individual CpG sites in enhancer elements. Methylation levels are shown for Tet2fl/fl;AE (Y-axis) and Tet2−/−;AE (X-axis) cultures at passage 2 (left panel) or passage 10 (right panel) after Tet2 disruption. CpG sites that are changing significantly [Q-value < 0.05; abs(diff) > 20%] are marked in red. (B) Cumulative distribution plot showing the methylation levels on all covered CpGs in enhancer elements. (Inset) Tet2-deficient cells have overall increased level of DNA methylation on enhancers upon cell passaging (p10 vs. p2 Tet2−/−;AE cells), indicating progressive hypermethylation. (C) Bar chart showing passage-dependent effects on DNA methylation in Tet2fl/fl;AE cells. Bars represent mean change of DNA methylation in the indicated genomic elements between passage 10 and passage 2. (D) Bar chart showing Tet2-dependent effects on DNA methylation at passage 2 (left panel) and passage 10 (right panel). Bars represent mean change of DNA methylation in the indicated genomic elements between Tet2−/−;AE (knockout) and Tet2fl/fl;AE (wild type). In both C and D, only CpG sites that showed significantly different DNA methylation levels [Q-value < 0.05; abs(diff) > 20%] were considered. The effect size of methylation change at each element was measured with Cohen's d. (*) Cohen's d between 0.5 and 1.0; (**) Cohen's d > 1.0. See Supplemental Figure S4 for details. (E) Serial replating assay of Tet2fl/fl or Tet2−/− Kit-enriched HSPCs transduced with MLL-AF9-expressing retrovirus. The cells were replated in methylcellulose-containing medium (M3534) every 5 d for a total of four replatings. Bars represent mean numbers of CFUs (n = 3), and error bars indicate SD. (F) Cumulative growth curve of Tet2fl/fl;MLL-AF9 or Tet2−/−;MLL-AF9 cultures (n = 3) grown for 10 passages. Cells were counted and plated at equal cell density every second day for a total of 20 d. (ns.) not significant (nonlinear regression). (G) Bar chart showing mean change of DNA methylation in the indicated genomic elements between Tet2−/−;MLL-AF9 (knockout) and Tet2fl/fl;MLL-AF9 (wild-type) cells 20 passages after Tet2 disruption. Mean changes of DNA methylation and genomic elements are presented in C and D. (*) Cohen's d between 0.5 and 1.0.

Figure 5.

Loss of 5hmC and gain of methylation lead to decreased H3K27ac and expression of neighboring genes. (A) Overview of 2107 regions in enhancers covered by eRRBS. The percentage of enhancer DMRs with an average DNA methylation increase (blue; mean difference >10%) or decrease (black; mean difference <−10%) are shown (left pie chart) as well as the fraction of hypermethylated enhancer DMRs associated with loss of 5hmC (log2 fold depletion >0.5) upon Tet2 knockout (right pie chart). See Supplemental Table S3 for full list of enhancer regions. (B) Quantitative bisulfite pyrosequencing validation of DNA methylation changes at four enhancer DMRs (Mtss1, Fam105a, Tmem123, and Las2 locus) associated with loss of 5hmC and gain of DNA methylation in Tet2−/−;AE cells. Bar graphs show methylation levels of each individual CpG within the locus in cells grown for two or 10 passages following Tet2 disruption. Bars represent mean methylation (n = 3), and error bars indicate SEM. (*) P-value < 0.05; (**) P-value < 0.01; (****) P-value > 0.0001 (two-way ANOVA). (C) Increase in DNA methylation levels at enhancer DMRs correlates with loss of H3K27ac, a surrogate marker of enhancer activity. Violin plots show the mean and lower and upper quartiles of the log2 transformed differential H3K27ac read density (knockout/wild type) for enhancer DMR + 250-bp flanking sequence. The 2107 enhancer DMRs have been divided into groups with <10% increase, 10%–30% increase, or >30% increase in DNA methylation levels upon Tet2 knockout. (*) P-value < 0.05; (****) P-value < 0.0001 (unpaired Student's t-test). (D) Increase in DNA methylation levels at enhancer DMRs correlates with decreased expression of genes within 100 kb. Violin plots show the mean and lower and upper quartiles of average z-scores (knockout/wild type) of genes within 100 kb of enhancer DMRs. Subgroups and significance levels are presented as in C. (E) Convergent gene regulation of AE and Tet2. Venn diagrams showing overlaps and corresponding P-values (Fisher's test, Yates-corrected) of genes deregulated in Tet2−/−;AE cells and genes bound by AE in AML patients (as described in Martens et al. 2012; Ptasinska et al. 2012). (Right panel) Heat map representing gene expression changes for genes differentially regulated by Tet2 knockout and bound by AE in both studies.

Figure 6.

Disruption of Tet2 leads to DNA hypermethylation of enhancers in murine and human AML cells. (A) Overview of the strategy for in vivo isolation of cells. GMP cells were sorted from the bone marrow of recipient mice 1 mo after transplantation of either wild-type or Tet2−/− bone marrow, AE-transduced cKit cells, or splenic cells from moribond Tet2−/−;AE leukemic mice (LeuA and LeuB) (see Fig. 1B). (B) DNA methylation levels assayed by quantitative bisulfite pyrosequencing at three enhancers (Mtss1, Tmem123, and Pde2a locus) in FACS-sorted wild-type (n = 4), Tet2−/− (n = 4), AE-transduced (n = 4), or leukemic Tet2−/−;AE (n = 8) GMP cells. Bars represent mean locus DNA methylation, and error bars show SD. (*) P-value < 0.05; (**) P-value < 0.01; (***) P-value < 0.001; (****) P-value < 0.0001 (unpaired Student's t-test). (C) Distribution of DNA methylation changes of significantly changing CpGs (P-value < 0.05 [Wilcoxon two-sample test, abs(diff) > 20%] in AML patients with TET2 mutations (left panel), t(8;21) translocation (middle panel), and DNMT3A mutations (right panel). The percentages of hypermethylated CpG sites versus all significantly changing sites are indicated on the plots. (D) Bar chart showing the distribution of the hypermethylated CpG sites in AML patients with TET2 mutation or t(8:21) translocation across various genomic elements. The number of hypermethylated CpG sites observed in each element was normalized to the number expected by random distribution. The P-value for enhancer CpGs in TET2 mutated patients is shown (Fisher's exact test, Yates correction). (DNMT3A mutated patients) Not sufficient hypermethylated sites for analysis. (E) As in C, but for probes hypomethylated in AML patients with DNMT3A mutations or t(8:21) translocation. (TET2 mutated patients) Not sufficient hypomethylated sites for analysis.

Figure 7.

Model of DNA methylation changes associated with aging and leukemogenesis. Normal hematopoietic cells experience age-dependent accumulation of DNA methylation on CpG islands (i). Mutation of TET2 leads to rapid hypermethylation of enhancers (ii) that, together with additional oncogenic aberrations (iii [as well as i]), can collaborate to induce malignant transformation.