Sirt1 Hmg20b Gm (0.17) 24 (17.3) 877 (857)
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1 3 (0.17) 24 (17.3) Sirt1 Hmg20 Gm (857) c d Suppl. Figure 1. Screen validation for top candidate antagonists of Dot1L (a) Numer of genes with one (gray), two (cyan) or three (red) shrna scored in the enriched 934 shrna pool from the genome-scale RNAi screen using the Dot1l-deletion model. Numers in parentheses indicate the expected numer of genes for each category ased on an average of 5 shrna per gene in the genome-scale lirary. Genes with three shrna scored in the screen are Sirt1, Hmg20 and Gm4763. Genes with more than 8 shrna in the lirary were excluded from the analysis. () Effect of EPZ4777 on the proliferation of murine MLL-AF9 leukemic cells transduced with the indicated shrna constructs. Cells were cultured in the presence of DMSO (open) or EPZ4777 (solid) for 9 days. (c) Frequency of the indicated sh-sirt1 in the genome-scale RNAi screen. (d) RT-qPCR of Sirt1 in MLL-AF9 leukemic cells transduced with indicated sh-sirt1 or sh-luc constructs. Data represent mean ± s.d. of a triplicated experiment. P < 0.05 to sh-luc. Suppl. Figure 2. Effect of Sirt1 knockdown and Dot1L inhiition on MLL-AF9 and AML1-ETO murine leukemic cells. Effect of Sirt1 knockdown on the proliferation of MLL-AF9 (left) and AML1-ETO (right) leukemic cells treated with DMSO or EPZ4777 (3 um) for 9 days. Cells were derived from retroviral-mediated transduction of MLL-AF9 or AML1-ETO fusion protein into mouse Lin - Sca1 + ckit + one marrow hematopoietic progenitors. Data represent mean ± s.d. of a triplicated experiment. P < 0.01.
2 Suppl. Figure 3. Retroviral-mediated overexpression of Meis1 or Hoxa7 increases the cell numer in MLL-AF9 cell cultures. (a) RT-qPCR of Meis1 (left) and Hoxa7 (right) in MLL-AF9 leukemic cells transduced with MSCV-puro viruses expressing Meis1 (red), Hoxa7 (lue), and vector control (green). Data represent mean ± s.d. of a duplicated experiment. P < () Effect of Meis1 or Hoxa7 overexpression on the cell numer of MLL-AF9 leukemic cells cultured for 9 days. Data represent mean ± s.d. of a triplicated experiment. P < Suppl. Figure 4. Validation of Sirt1 and Suv39h1 antiodies for immunoprecipitation. Lysate of murine MLL-AF9 leukemia cells was incuated with 10 ug/ml of anti-sirt1 (Acam; A12193; left panel), anti- Suv39h1 (Cell signaling; cat#8729; right panel) or normal IgG antiody overnight, and captured y protein A/G eads (Millipore) for 2 hours. The input, flow through (FT) and immunoprecipitated (IP) samples were then immunolotted for Sirt1 (left panel) and Suv39h1 (right panel).
3 c Suppl. Figure 5. Effect of Dot1L inhiition on Sirt1 and H3K79me2 levels in MLL-AF9 leukemic cells. (a) Semi-quantitative immunolot analysis for Sirt1 (Acam; A12193), H3K79me2 (Acam; A3594) and total histone H3 (Acam; A3594) levels in MLL-AF9 leukemic cells for (left) loading gradients (2 26 ul) of lysate from untreated cells, and (right) lysates (20 ul each) from cells treated with various concentrations of DOT1L inhiitor EPZ4777 (0 6 um). () Standard curves of Sirt1, H3K79me2 and total histone H3 derived from the loading gradients and densitometry analysis using ImageJ Software. (c) Relative levels of Sirt1 and H3K79me2 in MLL-AF9 leukemic cells treated with various concentrations of EPZ4777 were measured y densitometry (ImageJ software) and standard curve fitting. Sirt1 and H3K79me2 levels were normalized to the total histone H3 level of each sample. A nearly complete loss of H3K79me2, and a mild reduction (aout 30% less) of Sirt1 protein, was oserved in cells treated with more than 3 um EPZ4777 as compared to the DMSO control condition. Suppl. Figure 6. Dot1L inhiition does not alter the sucellular distriution of Sirt1 protein in MLL-AF9 leukemic cells. Immunofluorescence of MLL-AF9 leukemic cells cultured in DMSO versus EPZ4777 (3 um). Cells were stained with DAPI (lue; nucleus) and Sirt1 (red; A12193 from Acam). The white-dashed circles indicate the shape of the cells. DOT1L inhiitor treatment does not induce noticeale changes in the sucellular distriution of Sirt1 protein, which remains predominantly localized in the nucleus (overlaps with DAPI stain) of MLL-AF9 leukemic cells.
4 H3K79me2 H3K9ac Suppl. Figure 7. Meta analysis of ChIP-seq data from murine MLL-AF9 leukemia. Level (y-axis; reads per million) and position (x-axis; ± 10 k centered to TSS) of H3K79me2 and H3K9ac at highly expressed (red), not expressed (lue), MLL-AF9 ound target (yellow) and MLL-AF9 non-target control (gray; from Bernt et al Cancer Cell) genes. a Suppl. Figure 8. ChIP-seq analysis of H4K16ac in murine MLL-AF9 leukemia. (a) Meta analysis showing level (y-axis; reads per million) and position (x-axis; ± 10 k centered to TSS) of H4K16ac at highly expressed (red), not expressed (lue), MLL-AF9 ound target (yellow) and MLL-AF9 non-target control (gray; from Bernt et al Cancer Cell) genes. () Scatterplots showing changes in H3K9ac level (x-axis; Acam a4441) in correlation to changes in H4K16ac level (y-axis; Millipore ) at TSS ± 2 k regions in MLL-AF9 leukemic cells cultured in EPZ4777 (3 um) versus DMSO for 6 days. Gray dots represent all genes in the genome, and the MLL-AF9 targets in sh-luc (red dots) or sh-sirt1 (lue dots) transduced cells are highlighted.
5 Suppl. Figure 9. ATAC-seq chromatin accessiility profiles. Screen shots showing ATAC-seq profiles at MLL-AF9 ound target (Hoxa cluster and Meis1), active gene (Gapdh) and silent gene (H/Olf) loci in sh-luc (clone D11; green), sh-sirt1 (clone A1; red) and sh-suv39h1 (clone C2; lue) transduced MLL- AF9 leukemic cells cultured in DMSO or 1 um EPZ4777 for 6 days. The core occupied regions for MLL-AF9 are highlighted (red-dashed oxes), and the accessile chromatin regions ased on ATAC-seq signal are indicated (Peaks). Suppl. Figure 10. Knockdown of Sirt1 maintains chromatin accessiility at Hoxa7 and Meis1 upon deletion of Dot1l. To validate our ATAC-seq transposase accessiility assay (shown in Fig. 5i), we used nuclease digestion of chromatin and qpcr to detect chromatin accessiility at Hoxa7 and Meis1 loci in sh-luc (clone D11) or sh-sirt1 (clone A1) transduced MLL- AF9-Dot1l f/f -CreER leukemic cells under control and Dot1l-excised (4OHT) conditions. Samples were processed and analyzed using EpiQ Chromatin Analysis Kit according to the manufacturer s protocol (Bio-Rad). The Gapdh locus served as a positive control gene for accessile chromatin. Data represent mean ± s.d. of two experiments. P < 0.05.
6 DOT1L DOT1L-deletion or inhiition DOT1L MLL SEC Active MLLAF9 targeted genes MLL SEC Silent X MLLAF9 targeted genes H3K79me2 H3K9ac H3K79me2 H3K9ac SIRT1 SUV39H1 SIRT1 SUV39H1 Suppl. Figure 11. Model of Regulation of MLL-AF9 Target Genes y DOT1L, SIRT1 and SUV39H1 in MLL-AF9 leukemia. (a) Proposed model for MLL-AF9/DOT1L-driven gene expression in MLL-AF9 leukemia. SEC: super elongation complex. () Involvement of SIRT1/SUV39H1-mediated repressive mechanisms in silencing MLL-AF9 target gene expression after suppression of DOT1L. Suppl. Figure 12. SRT1720 treatment alone induces limited effect on the cell numer of human leukemic cell cultures. Effect of SRT1720 (1uM) on the proliferation of human MLL-rearranged Molm13 (MLL-AF9), MV4-11 (MLL-AF4) and SEMK2 (MLL-AF4); and non-mll-rearranged Kasumi-1 (AML-ETO) and HL-60 leukemic cell lines cultured for 17 days. Data represent mean ± s.d. of a duplicated experiment.
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