administration of tamoxifen. Bars show mean ± s.e.m (n=10-11). P-value was determined by

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1 Supplementary Figure 1. Chimerism of CD GFP + cells at 1 month post transplantation No significant changes were detected in chimerism of CD GFP + cells between recipient mice repopulated with Ezh2 wt/wt and Ezh2 flox/flox HSCs at 1 month post transplantation, prior to the administration of tamoxifen. Bars show mean ± s.e.m (n=10-11). P-value was determined by Student s t-test.

2 Supplementary Figure 2. Successful deletion of Ezh2 alleles Genomic PCR utilizing CD donor-derived BM cells showed successful deletion of Ezh2 alleles in Ezh2 Δ/Δ and Rx291/Ezh2 Δ/Δ mice. The PCR primers are shown in Supplementary Table 4, and standard PCR with Taq DNA polymerase was performed in condition of (94 C 30sec/60 C 30sec/72 C 2min) x 35 cycles.

3 Supplementary Figure 3. Detection of human RUNX1 transcript No significant change was detected in the levels of RUNX1S291fs transcripts between Rx291 LSK and Rx291/Ezh2 Δ/Δ LSK cells by quantitative PCR utilizing primers specific to human RUNX1 transcript. Bars show mean ± s.d. (n=4-5). P-value was determined by Student s t-test.

4 Supplementary Figure 4. GSEA plots for Ezh2 targets and HSCs fingerprints genes Enrichment plots of GSEA are shown for Ezh2 targets (Shen E, et al. Mol Cell 2008) and HSCs fingerprints (Chambers SM, et al. Cell Stem Cell 2007). Genes were examined in CD GFP + LSK cells from Ezh2 Δ/Δ, Rx291 and Rx291/Ezh2 Δ/Δ mice at pre MDS stage and Rx291/Ezh2 Δ/Δ MDS and MDS/ML mice, compared to those from WT mice.

5 Supplementary Figure 5. Colony-forming capacities of CD LSK cells Colony-forming capacities were examined in CD LSK cells isolated from Rx291/Ezh2 Δ/Δ mice, CD LSK cells isolated from Rx291 mice, and the primary wild-type LSK cells. Proliferative capacity was significantly impaired in CD wild-type LSK cells isolated from Rx291/Ezh2 Δ/Δ mice. Bars and asterisk show mean ± s.e.m., **p<0.01, and ***p<0.001 by Student s t-test (n=3-4).

6 Supplementary Figure 6. GSEA plots for the IL-6 and TNF pathway genes in Rx291 mice Gene set enrichment plots for the IL-6 (left panel) and TNF (right panel) pathway gene sets, comparing CD wild-type LSK cells to CD GFP + mutant LSK cells in Rx291 mice. Neither of pathways were upregulated in CD wild-type LSK cells in Rx291 mice.

7 Supplementary Figure 7. GSEA plots for AML target genes Enrichment plots of GSEA are shown for MLL-AF9 (Krivtsov AV, et al. Nature 2006) and NUP98-HOXA9 (Takeda A, et al. Cancer Res 2006) targets. Genes were examined in CD GFP + LSK cells from Ezh2 Δ/Δ, Rx291 and Rx291/Ezh2 Δ/Δ mice at pre MDS stage and Rx291/Ezh2 Δ/Δ MDS and MDS/ML mice, compared to those from WT mice. Rx291/Ezh2 Δ/Δ -MDS LSK cells had negative enrichment of canonical AML target genes.

8 Supplementary Figure 8. RRBS analysis at Hoxa cluster regions No differential DNA methylation was seen at the promoter region of Hoxa9 between wild-type, Rx291, Rx291/Ezh2 Δ/Δ and Rx291/Ezh2 Δ/Δ -MDS LSK cells. The percentages of DNA methylation at the Hoxa cluster regions defined by RRBS are depicted in these LSK cells.

9 Supplementary Figure 9. RUNX1S291fs mutant associates with Bmi1 in vivo While it is hard to evaluate co-immunoprecipitation of wild-type Runx1 with Bmi1 due to the overlapping Ig heavy chain bands, RUNX1S291fs mutant protein was obviously co-immunoprecipitated with Bmi1 using an anti-bmi1 antibody, but not pre-immune IgG. Bmi1 protein was hardly detected in the input lanes under this condition of experiment.

10 Supplementary Figure 10. H3K27me3 and H2AK119ub1 levels at Hoxa9 in Ezh2 Δ/Δ cells ChIP analysis utilizing either H3K27me3 or H2AK119ub1 antibody was performed in (CD GFP + ) WT and Ezh2 Δ/Δ LSK cells. While Ezh2 loss alone did not significantly affect the level of H3K27me3 at the promoter of Hoxa9, it induced a mild but not significant increase in the level of H2AK119ub1. Bars show mean ± s.e.m (n=4). P-value was determined by Student s t-test.

11 Supplementary Figure 11 Full blots presented in the figures.

12 Supplementary Table 1. Phenotypic features of Rx291-MDS and Rx291/Ezh2 Δ/Δ -MDS mice Diagnosis WBC Hb (g/dl) neutrophils in PB monocytes in PB MCV PLT (x10 4 /ul) BM (x10 6 ) Myeloblasts in BM (%) Spleen (mg) Rx291-MDS 1 MDS MDS Rx291/Ezh2 Δ/Δ -MDS 1 MDS MDS MDS n.a MDS/ML MDS MDS MDS MDS MDS n.a MDS MDS n.a MDS MDS Rx291/Ezh2 Δ/Δ (n=17) pre-mds 13500± ± ± ± ± ± ± ±0.3 80±29

13 Supplementary Table 2. List of DNA hypermethylated canonical Ezh2 target genes Ezh2 targets in Rx291/Ezh2 Δ/Δ hypermethylated gene Adamts18 Artn Bcor Boll Dcc Drd2 Fbxo32 Gabrg3 Gnai1 Papolb Pcdh9 Pdx1 Rasef Slc7a2 St6gal2 Xkr4 Ezh2 targets in Rx291/Ezh2 Δ/Δ -MDS hypermethylated gene Alox5 Bai3 Cdh13 Col8a2 Fbxo32 Gabrg3 Hand1 Il11 Mal2 Mapk13 Papolb Pdx1 Rasef Slc34a2 Sprn Sstr4

14 Supplementary Table 3. GSEA for inflammatory cytokine responses GSEA for the interferon- (Reactome), TNF (TNF receptor signaling, Pathway Interaction Database), and IL-6 (Dasu MRK, et al. J Pathol 2004) pathway gene sets in CD wild-type LSK cells and CD GFP + mutant LSK cells from Rx291 and Rx291/Ezh2 Δ/Δ mice at 4 months post transplantation (pre-mds stage) compared to LSK cells from WT mice. NES and FDR q-value are shown as in Table 2. mice Rx291 Rx291 Rx291/Ezh2 Δ/Δ Rx291/Ezh2 Δ/Δ Genotype Gene set CD GFP + Rx291 CD wild type CD GFP + Rx291/Ezh2 Δ/Δ CD wild type IFNg pathway 0.67/ / / /0.173 IL-6 pathway 1.52/ / / /0.009 TNFa pathway 0.93/ / / /0.506

15 Supplementary Table 4. Primer information RT-PCR Gene Forward primer Reverse primer Roche-Universal Probe Library Bmi1 CAAAACCAGACCACTCCTGAA TCTTCTTCTCTTCATCTCATTTTTGA No 95 Hoxa9 AGGCAAACGAATCTGTTGGT TGTTTCGGAAGCCACACA No 31 IL-6 TCTAATTCATATCTTCAACCAAGAGG TGGTCCTTAGCCACTCCTTC No 78 Mecom/Evi1 GGATGACTACGAAGAAGCTGGT AGAGCAGAAAGGCCAGATTTATAG No 80 RUNX1 ATGAGGGTCAGCCCACAC GATGGTTGGATCTGCCTTGTA No 23 RT-PCR Gene Forward primer Reverse primer Hoxa10 CCTTCAGAAAACAGTAAAGCTTCG AAGGGCAGCGTTTCTTCC Gapdh ATGACATCAAGAAGGTGGTGAAG TCCTTGGAGGCCATGTAGG ChIP-qPCR Gene Forward primer Reverse primer Hoxa2 GACAAGGTTGAAATTGGACCG CAAATTGTCATTGGGCAGAAGC Hoxa6 CTTTCCTTTTTTGCCTTCATGG TTGTCAGGTTTCCTGTTTGGG Hoxa9 TCACCACCACCCCTACGT GCAAGCCCGCGAAGGA Hoxa10 CTGGCTCTTGAACCTGTACCCC CAAGGGTGCTTCCAAATAGTC Hoxa11 GGAAGCAACAGATCGTCACTCG TGAGTTACACCGGCGATTACG Mecom/Evi1, promoter ACGCACACACTCGGTCTTTCACTC GTAGCTCCCTTTCTCCGCCTCCT Mecom/Evi1, intron GTACCACCCACATTTCTTTCTCTC CCAAAATGAATTAGTCACCACCTC Genotype PCR Gene Forward primer Reverse primer Ezh2 AAGGCTGTGTACAGGAAACAATC ATGCATCTGTCACTGGTAACCTAC