DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells

Abstract

Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation-hydroxymethylation pathway. Using Dnmt3a and Tet2 double-knockout mice in which the development of malignancy is accelerated, we show that the double-knockout methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulators Klf1 and Epor, is upregulated in double-knockout hematopoietic stem cells (HSCs). DNMT3A and TET2 both repress Klf1, suggesting a model of cooperative inhibition by epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, the loss of which, along with DNMT3A, obstructs differentiation, leading to transformation.

Figure 1. Phenotype of Dnmt3a-Tet2 DKO mice

a) Engraftment of donor-derived bone marrow cells after competitive bone marrow transplantation (BMT) of wild type, Dnmt3a^−/−, Tet2^−/− and DKO in WBC, myeloid, B cell and T cell compartments in the first 12 weeks. n=15 for each group. b) Kaplan-Meier survival of BMT recipients ***, p<0.001, Log-rank test. c) Complete blood counts of BMT recipients after 4 months. WBC, white blood cell. RBC, Red blood cell, MCV, Mean corpuscular volume. n=10 for each group. d–e) Donor-derived cells stained with myeloid markers Mac1 and Gr1 6 months after BMT. f–g) Donor-derived cells stained with the erythroid markers CD71 and Ter119 6 months after BMT. n=3 for each group. h) Hematopoietic progenitor analysis (Lineage^neg Scal⁺cKit⁺) in representative mice 1 month after Poly(I:C) injection; (i) Quantification of myeloid progenitors (LK) in donor-derived cells from (h) n=3 for each group. (j,k) Percentage of hematopoietic progenitors (LSK) in donor-derived cells 4 months after BMT. n=3 for each group. All error bars show the mean±S.E.M. n.s, not significant; * p<0.05; ** p<0.01. Two tailed student T test.

Figure 2. Synergistic dysregulation of HSC- and RBC-associated genes in DKO HSCs

a) Heatmap displaying all differentially expressed genes in each genotype relative to WT. b) Plot representing GSEA enrichment of Fingerprint lineage-specific genes among differentially expressed genes between the indicated genotypes. Normalized enrichment scores are plotted with FDR q value. HSC: Hematopoietic Stem Cell. NuRBC: nucleated Red Blood Cell. c) Expression levels of the indicated genes in HSCs. p<0.05. **, p<0.01. ***, p<0.001. Each group consist of 2 biological replicates. p value was calculated by cuffdiff in the RNA-seq analysis pipeline. d) Functional pathway enrichment analysis of genes up-regulated in DKO vs. Tet2^−/− HSCs. e) Venn diagram depicting the overlap between genes upregulated in DKO vs. Tet2^−/− HSCs compared with activated and repressed KLF1 targets. **, p<0.01. Fisher exact test. The indicated genes are members of the heme synthesis pathway. f) Box plot depicting the range of expression of KLF1 target genes from (e) which are activated in the DKO relative to the Tet2^−/−. Boxplots represent the interquartile range (25% to 75%), with the median; whiskers correspond to 1.5 times the interquartile range. Each group consist of 2 biological replicates. Each * p<0.05. **, p<0.01. ns, not significant. p value was calculated by cuffdiff in RNA-seq analysis pipeline.

Figure 3. Knockdown of Klf1 and Epor reverses the abnormal self-renewal of DKO HSPCs in vitro

a) Replating assay with DKO Lin− cKit+ Sca1+ (LSK) HSPC cells transfected with shScramble, shEpor, and shKlf1 using M3434 methylcellulose medium. n=3 for each treatment group. b) ckit expression on plated DKO LSK HSPC cells transfected with shScramble, shEpor, and shKlf1 cells. c) Immunoblot shows the knockdown of Epor and the expression level of downstream targets of Epor after knockdown of Epor in DKO HSPCs. d) Replating assay with DKO whole bone marrow cell after treatment with the JAK2 inhibitor Ruxolitinib and Bcl-xL specific inhibitor WEHI-569. n=3 for each treatment group. e) ckit expression on plated DKO whole bone marrow cells treated with Jak2 inhibitor Ruxolitinib and Bcl-xL specific inhibitor WEHI-569. All error bars show the mean ± S.E.M. *, p<0.05, Student T-test.

Figure 4. DNA methylation across the genotypes are highly dynamic

a) Flowchart of DNA methylation analysis strategy. b) Heat map depicting differentially methylated regions (DMRs) of the 6 major dynamic DNA methylation alteration patterns (DMAPs). c) Numbers of hypermethylated and hypomethylated DMRs in HSCs of Dnmt3a^−/−, Tet2^−/− and DKO phenotype. d) Global DNA methylation levels of all DMRs in all 4 genotypes. Each group of genotype consisted of 2 biological replicates. e) DNA methylation levels in hematopoiesis-associated enhancers in different lineages and progenitors. Enhancers (left panel) as defined by H3K27Ac marks in B cell, RBC progenitors, ST- and LT-HSCs (from ref. 43), and (right panel) their DNA methylation levels.

Figure 5. Hydroxymethylation (hmC) in HSCs is associated with active HSC genes and repressed RBC genes

a) Numbers of hypermethylated and hypomethylated DhMRs in HSCs of Dnmt3a^−/−, Tet2^−/− and DKO phenotype. b) Heat map depicting differentially hydroxylmethylated regions (DhMRs) of the 5 major dynamic DNA hydroxylmethylation alteration patterns (DhMAPs). c) Cytosine-5-methylenesulfonate (CMS) signal in HSCs displayed for DMR regions. d) CMS hmC signal in displayed in relationship to genic features. e–h) Differential CMS signal distribution in regions within the 6 major DMR classes in HSCs.

Figure 6. Hydroxymethylation (hmC) in HSCs is associated with active HSC genes and repressed RBC genes

a) Overlap between DMRs and DhMRs in Dnmt3a^−/−, Tet2^−/− and DKO HSCs. p value is calculated with Chi-square test. b) Relative overlap ratio of 6 different dynamic DMR patterns with 5 clusters of DhMRs in comparison with random overlap of DMR and DhMRs. The grey line indicates random DMR and DhMR global overlap ratio. Observed/Expected is plotted. c) Distribution of mC in genic regions of genes from low to high expression level in HSCs of all 4 genotypes. TSS: transcriptional start site. TTS: transcriptional termination site. d) Distribution of mC in genic regions of genes from low to high expression level in HSCs of all 4 genotypes. TSS: transcriptional start site. TTS: transcriptional termination site. e) Gene expression level (FPKM) of HSC Fingerprint genes (upper panel) analyzed in f and RBC Fingerprint genes (lower panel) analyzed in g. Each genotype group consisted of 2 biological replicates. f) hmC signal distribution on HSC-Fingerprint genes down-regulated in DKO HSCs. Each genotype group consisted of 2 biological replicates. g) hmC signal on RBC-Fingerprint genes associated with up-regulation in DKO HSCs. Each genotype consisted of 2 biological replicates. h) The hmC signal distribution of the blue shaded area from (g) containing the TSS±200bp region *, p<0.05. Student t-test.

Figure 7. DNMT3A and TET2 cooperate to prevent activation of lineage-specific transcription factors in HSCs

a) Comprehensive epigenomic dynamics at the Mpl locus in HSCs with tracks WGBS, CMS, H3K27me3 and gene expression (RNAseq). Blue shaded area shows the cluster 5 DhMR located in the gene body of Mpl. b) Comprehensive epigenomic dynamics at the Klf1 locus in HSCs. Blue shaded area shows the Type III DMR located in the promoter and 5′UTR regions of Klf1. Red shaded area shows the Cluster 4 DhMR located in the TSS and 5′UTR region in which hmC increases in Dnmt3a^−/− HSC and decrease in Tet2^−/− and DKO HSCs. The y axis of CMS and H3K27me3 sections shows signal of ChIP-seq occupancy in units of reads per million mapped reads per base pair (rpm/bp) in a and b. c, d) Model shows the action mode of DNMT3A and TET2 in activating HSC gene expression and repressing lineage-specific transcription factor expression.