The Biological Function of DNA Hypermethylation in Murine MDS Model Lacking Tet2 and Ezh2

Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2^KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2^fl/fl)(Muto T, et al. J Exp Med 2014).

In order to determine how DNA methylation contributes to the formation of MDS in Tet2^KD/KDEzh2^Δ/Δ mice, we transplanted wild type (WT), Tet2^KD/KD, Cre-ERT;Ezh2^fl/fl, and Cre-ERT;Tet2^KD/KDEzh2^fl/fl fetal liver cells in lethally irradiated CD45.1⁺ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin^-Sca1⁺Kit⁺ (LSK) cells isolated from Tet2^KD/KD and Ezh2^Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2^KD/KDEzh2^Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value<0.01). We found that Ezh2^Δ/Δ LSK cells gain significantly larger number of hyper-DMRs at CpG islands (CGIs) over time compared with Tet2^KD/KD LSK cells. At 8 months post-deletion, Tet2^KD/KD and Ezh2^Δ/Δ LSK cells gained hyper-DMRs in 196 and 366 out of 12677 promoters, and 106 and 306 out of 11504 CGIs, respectively. Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells at 5 months post-deletion had hyper- and hypo-DMRs in 109 and 51 out of 12677 promoters, and 124 and 27 out of 11504 CGIs, respectively. Hyper-CGI DMRs of Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells largely overlapped with those of Tet2^KD/KD and Ezh2^Δ/Δ LSK cells (p<1.00E-16). Nonetheless, a significant number of hyper-CGI DMRs were newly generated in Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells, suggesting unique impact of combined loss of Tet2 and Ezh2 on epigenome. Of note, hyper-DMRs in this mouse MDS model significantly overlapped with those in human CD34⁺ cells of MDS patients (Maegawa S, et al. Genome Research 2014) (40 genes, p=0.0001). As reported in solid tumor and aging stem cells, polycomb-group (PcG) protein targets were significantly enriched among hyper-CGI DMRs in LSK cells of all three genotypes; Tet2^KD/KD, Ezh2^Δ/Δ, and Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells. Indeed, hypermethylated CGIs were enriched for genes encoding DNA binding transcription factors (p=8.96E-05) and cell differentiation (p=3.98E-05) in GO analysis, suggesting that DNA methylation involves the PcG targets such as developmental regulator genes that regulate hematopoietic cell differentiation in the pathogenesis of MDS.

To determine an association between levels of differential DNA methylation and transcription of genes, we next performed DNA microarray analysis by using LSK cells. As several studies reported, we observed no significant association between levels of DNA methylation and gene expression (R²=0.003). However, 27 hypermethylated genes showed reduced levels of gene expression in Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells. Those included key transcription factors and epigenetic modifiers for hematopoiesis, such as Gata2, Gata3, Evi1, and Bcor.

Finally, we examined whether decitabine, a demethylating agent, reverses the DNA hypermethylation of Tet2^KD/KDEzh2^Δ/Δ-MDS LSK cells in vivo. Although the administration of decitabine into secondary recipients of MDS BM cells did not improve the survival of mice, platelet counts were significantly increased in decitabine-treated MDS mice, compared to the control MDS mice. After 4 weeks of treatment, a partial restoration of DNA hypermethylation was observed in the decitabine-treated MDS mice (34 out of 124 CGIs; p<1.00-16E). These regions were again enriched in DNA binding transcription factors (p=1.72E-05) and cell differentiation (p=2.25E-07) in GO analysis.

Taken together, our work unveiled the biological function of DNA hypermethylation in the pathogenesis of MDS, and will give a clue to understanding how demethylating agents improve the outcome of patients with MDS.