Schematic overview of studies of DNA methylation and RNA expression by zebrafish kidney marrow cells. The site of hematopoiesis in the adult zebrafish is in the head kidney (upper left of the diagram). Head kidney marrow cells from 4- and 15-month-old zebrafish were isolated and the progenitor cells were sorted by fluorescence-activated cell sorting based on their forward and side light scatter properties, as previously described (7). The sorted progenitor cells were then processed for DNA extraction using enhanced reduced representation of bisulfite sequencing to assess cytosine methylation. RNA was also extracted from an aliquot of the sorted progenitor cells for poly(A)+ RNA sequencing, as described in the Methods section. tet2, Ten-eleven translocation 2; ERRBS, Enhanced Reduced Representation of Bisulfite sequencing; Ey, erythrocytes; Ly, lymphocytes; Pr, progenitors; My, myelomonocytes.

Loss of tet2 associated with the gain of CpG methylation in HSPCs of 4-month-old. (A) Principal component analysis (PCA) of high-variance CpGs showing the variability between biological samples and similarities between the replicates (the replicates are indicated as R1 and R2). (B) Density plots showing the distribution of the percent DNA CpG methylation in HSPCs of 4-month-old tet2^m/m and 4-month-old tet2^wt/wt fish. The difference between the distribution curves was calculated using the Wilcoxon rank-sum test. (C) The percentage differences in the methylation of cytosines within the promoter regions (500 bp to +250 bp upstream and downstream of the transcription initiation site) of annotated genes from 4-month-old tet2^m/m fish compared with 4-month-old tet2^wt/wt fish. The percentage difference in the methylation status of CpG sites in each promoter region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The promoters with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. Genes that were hypermethylated in the 4-month-old tet2^m/m fish are colored in orange and those that were hypomethylated are colored in blue. (D) Gene set enrichment analysis of hypermethylated gene promoters (human orthologs) against the transcription factor target ChEA 2022 database using EnrichR. The five most significant pathways are shown. The x-axis indicates the negative logarithm of the p-value from the enrichment results. (E) Gene set enrichment analysis of hypomethylated gene promoters (human orthologs) against the transcription factor target (ChEA 2022) database using EnrichR, as in panel (C) The five most significant pathways were plotted. The x-axis shows the negative logarithm of the p-value from the enrichment results. (F) The percentage differences in the methylation of cytosines within the CpG island regions in 4-month-old tet2^m/m fish compared with 4-month-old tet2^wt/wt fish. The percentage difference in the methylation status of CpG sites in each CpG island region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The CpG islands with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. The CpG islands that were hypermethylated in the 4-month-old tet2^m/m fish are colored in orange and those that were hypomethylated are colored in blue.

DNA methylation changes in HSPCs of aging tet2^m/m fish. (A) The percentage differences in the methylation of cytosines within the promoter regions (500 bp to +250 bp upstream and downstream of the transcription initiation site) of annotated genes from 15-month-old tet2^m/m fish compared with 4-month-old tet2^m/m fish. The percentage difference in the methylation status of each promoter region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The regions with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. The genes that were hypermethylated in the 15-month-old tet2^m/m fish are colored in orange and those that were hypomethylated are colored in blue. (B) Gene set enrichment analysis of hypermethylated gene promoters (human orthologs) against the ChEA 2022 database using EnrichR. The top five significant pathways were plotted. The x-axis indicates the negative logarithm of the p-value from the enrichment results. The y-axis represents individual pathways. (C) Gene set enrichment analysis of hypomethylated gene promoters (human orthologs) against the transcription factor target database (ChEA 2022) using EnrichR. The top five significant pathways were plotted. The x-axis indicates the negative logarithm of the p-value from the enrichment results. The y-axis indicates the individual pathways. (D) The percentage differences in the methylation of cytosines within the CpG island regions in 15-month-old tet2^m/m fish compared with 4-month-old tet2^m/m fish. The percentage difference in methylation status of CpG sites in each CpG island region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The CpG islands with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. The CpG islands that were hypermethylated in the 4-month-old tet2^m/m fish are colored in orange and those that were hypomethylated are colored in blue.

DNA methylation changes in HSPCs of aging tet2^wt/wt fish. (A) The percentage differences in the methylation of cytosines within the promoter regions (500 bp to +250 bp upstream and downstream of the transcription initiation site). HSPCs from 15-month-old tet2^wt/wt fish were compared with those of 4-month-old tet2^wt/wt fish, with the latter as the baseline. The percentage difference in the methylation status of CpG sites in each promoter region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The regions with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. The genes that were hypermethylated in the 15-month-old tet2^wt/wt fish are colored in orange and those that were hypomethylated are colored in blue. (B) Horizontal bar plot comparing the methylation status of significantly differentially methylated promoters identified in 15-month-old tet2^m/m fish compared with 4-month-old tet2^m/m fish (top); 4-month-old tet2^m/m fish compared with 4-month-old tet2^wt/wt fish (middle); and 15-month-old tet2^wt/wt fish compared with 4-month-old tet2^wt/wt fish (bottom). Orange represents > 15% hypermethylation, blue represents > 15% hypomethylation, and white represents no differential methylation.

Overlap of DNA CpG promoter methylation in tet2^m/m fish with changes previously reported in human MDS specimens. (A) The percentage differences in the methylation of cytosines within the promoter regions (500 bp to +250 bp upstream and downstream of the transcription initiation site) in the HSPCs of 15-month-old tet2^m/m fish compared with those 15-month-old tet2^wt/wt fish, with the latter as the baseline. The percentage difference in the methylation status of the CpG sites in each promoter region is plotted on the x-axis and the negative logarithm of the adjusted p-value is on the y-axis. The regions with an absolute change greater than 15% and an adjusted p-value less than 0.05 are colored. The genes that were hypermethylated in the 15-month-old tet2^m/m fish are colored in orange and those that were hypomethylated are colored in blue. (B) Overlap of differentially methylated promoters identified in Figure 5A with those identified in human MDS specimens. Common genes are highlighted—genes in orange are hypermethylated in the HSPCs of 15-month-old tet2^m/m fish and those in blue are hypomethylated in the HSPCs of 15-month-old tet2^m/m fish.

Loss of tet2 associated with changes in expression of genes associated with inflammation, metabolism, and the cell cycle in 4-month HSPCs. (A) Volcano plot representing the differentially expressed genes identified from the bulk-RNAseq sequencing data. Differentially expressed genes were identified between 4-month-old tet2^m/m and 4-month-old tet2^wt/wt HSPCs, with 4-month-old tet2^wt/wt as the baseline. The genes with an absolute log2-fold change greater than 1 and an adjusted p-value less than 0.05 are colored. The upregulated genes in the 4-month-old tet2^m/m fish are colored in violet and the downregulated genes in the 4-month-old tet2^wt/wt fish are colored in dark green. (B) Pathway enrichment analysis of the upregulated genes against the hallmark database was conducted using EnrichR. The top five significant pathways (p < 0.05) were plotted. The x-axis indicates the negative logarithm of the p-value from the enrichment results. The y-axis represents individual pathways. (C) Pathway enrichment analysis of the downregulated genes against the hallmark database was conducted using EnrichR. The top five significant pathways were plotted. The x-axis indicates the negative logarithm of the p-value from the enrichment results. The y-axis represents individual pathways.

Upregulation of interferon-induced protein 44-like (IFI44L). Bar plots representing the normalized expression values averaged across replicates on the y-axis and 4-month-old tet2^wt/wt fish and tet2^m/m fish on the x-axis. The error bars are calculated as mean expression ± standard deviation. (A–D) represent different orthologs of this gene in zebrafish.

Representative downregulated genes associated with the heme metabolism pathway. Bar plots representing the normalized expression values averaged across replicates on the y-axis and 4-month-old tet2^wt/wt fish and tet2^m/m fish on the x-axis. The error bars are calculated as mean expression ± standard deviation. PIGQ, Phosphatidylinositol Glycan Anchor Biosynthesis Class Q; SLC4A1, Solute Carrier Family 4 Member 1; CPOX, Coproporphyrinogen Oxidase.

Negative association between hypermethylated gene promoters and gene expression changes observed in 4-month-old tet2^m/m fish compared with 4-month-old tet2^wt/wt fish. Gene set enrichment analysis of hypermethylated promoters identified in Figure 2A against the ranked list of genes (sorted from the highest fold change value to the lowest) identified from Figure 6A.