Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome

INTRODUCTION: Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2(m/m)-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. METHODS: In the current study, we collected progenitor cells from the kidney marrows of the adult tet2(m/m) and tet2(wt/wt) fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). RESULTS AND DISCUSSION: A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2(m/m) HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)—involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2(m/m) HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2(m/m) HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.