Article Figures & Data
Figures
- Figure 1.
Structure and binding properties of the CBX8–AF9 AHD complex. A, Ensemble of the 10 lowest energy conformers from the structure calculations with CBX8 in orange and AF9 in purple. B, Alignment of the CBX8–AF9 and DOT1L–AF9 (PDB: 2MV7) structures with CBX8 in orange, its AF9 in purple, DOT1L in gold, and its AF9 in cyan. Backbone RMSD for AF9 residues 502–562 between CBX8–AF9 and the other AF9 complexes is shown in the table on the right. See also Supplementary Table S1 and Supplementary Fig. S1. C, Results of FA assays for binding of MBP-AF9 to fluorescein-labeled CBX8 WT (blue) and A335V (black) peptides. Error bars indicate SEM for three replicates. D, Top, sequence alignment of the AF9 binding motif. Conserved hydrophobic residues are in red. Bottom-comparison of the binding affinities of fluoresceinated peptides for all binding partners for MBP-ENL to affinities for MBP-AF9 measured by FA. Fold change is calculated as AF9 KD/ENL KD. Error bars, SEM for three replicates. E, Results of FA assays for competition of a fluorescein-labeled DOT1L site 3 peptide off of MBP-AF9 by unlabeled CBX8 A335V (black), WT (blue), and R336D (red) peptides. Error bars, SEM for three replicates. For details, see Supplementary Fig. S2.
- Figure 2.
CBX8 A335V inhibits colony formation, and CBX8 R336D has no effect. MLL-AF9–transformed mouse BM cells from Cbx8fl/fl (flox/flox) and conditional Cbx8 knockout (Cbx8KO) mice after methylcellulose culture 7 days with vehicle or 4-hydroxytamoxifen (4-OHT). For A, B, D, and E, data are from two experiments, each in triplicate, and are represented as mean ± SEM. Statistical significance was determined using a Student two-tailed t test with a confidence interval of 95%. A, Quantification of endogenous Cbx8 RNA expression, normalized to Polr2a by qRT-PCR. B, Quantification of colony and cell number relative to vehicle control. C, Bright-field images of colonies treated with vehicle (top) or 4-OHT (bottom). D, Quantification of exogenous CBX8 WT (gray), A335V (red), and R336D (blue) RNA expression, normalized to Polr2a by qRT-PCR. E, Number of colonies (panels 1 and 2) and cells (panels 3 and 4) produced by conditional Cbx8 knockout mouse BM cells treated with 4-OHT (panels 1 and 3) or vehicle (panels 2 and 4) and expressing exogenous CBX8 WT (gray), A335V (red), or R336D (blue). F, Bright-field images of conditional Cbx8 knockout mouse BM cells expressing CBX8 WT (left), A335V (middle), or R336D (right) and treated with vehicle (top) or 4-OHT (bottom). G, Images of Wright–Giemsa stained flox/flox (top) and conditional Cbx8 knockout (bottom) mouse BM cells treated with vehicle (panel 1) or 4-OHT and expressing exogenous CBX8 WT (panel 2), A335V (panel 3), or R336D (panel 4).
- Figure 3.
Structure and binding properties of the BCOR–AF9 AHD complex. A, Ensemble of the 10 lowest energy conformers from the structure calculations, with BCOR in red and AF9 in blue. Unstructured BCOR residues 1176–1191 have been omitted for clarity. B, Alignment of the BCOR–AF9 and DOT1L–AF9 structures, with BCOR in red and its AF9 in blue, and DOT1L in gold and its AF9 in cyan. Backbone RMSD for AF9 residues 502–562 between BCOR–AF9 and the other AF9 complexes is shown in the table on the right. Unstructured BCOR residues 1176–1191 and DOT1L residues 893–900 have been omitted for clarity. See also Supplementary Table S2 and Supplementary Fig. S1. C, Results of FA assays for binding of MBP-AF9 to fluorescein-labeled BCOR (1176–1206) (blue) and BCOR (1176–1225) (black) peptides. Error bars, SEM for three replicates. D, Weighted chemical shift difference for AF9 residues in 15N-1H HSQC spectra of BCOR (1176–1228)–AF9 versus BCOR (1175–1207)–AF9 complexes calculated using |Δδ15N|/4.69 + |Δδ1HN|. AF9 E531 is shown in red. E, Top, results of FA assays for binding of MBP-AF9 E531R (blue) and MBP-AF9 (black) to a fluorescein-labeled BCOR (1176–1225) peptide. Error bars, SEM for three replicates. Bottom, effect of the AF9 E531R mutation on the binding of fluoresceinated peptides of all binding partners measured by FA. Fold change is calculated as E531R KD/WT KD. See also Supplementary Fig. S2. F, Immunoprecipitation (IP) of endogenous BCOR with FLAG-tagged AF9 or AF9 E531R from HEK293T cells. One representative immunoblot and summary from three independent experiments quantified relative to input MLLT3 levels.
- Figure 4.
MLL-AF9 E531R shows increased cell proliferation and prevents leukemogenesis in vivo. A, Quantification of methylcellulose colony formation from serial replating of MLL-AF9– and MLL-AF9 E531R– or D546R–transduced mouse BM cells. Data are from six experiments, each in triplicate, and are represented as mean ± SEM. B, Bright-field images of MLL-AF9 WT and E531R colonies. C, Quantification of cells per colony formed from serial replating of transduced mouse BM cells as in A. Data are from six experiments, each in triplicate, and are represented as mean ± SEM. Statistical significance was determined using a Student two-tailed t test with a confidence interval of 95%. D, Feret diameter box plots for MLL-AF9 WT (n = 115) and MLL-AF9 E531R (n = 88) colonies. Box indicates 1st and 3rd quartiles, line indicates median, and whiskers represent max and min. Statistical significance was determined using a Student two-tailed t test with a confidence interval of 95%. E, Images of Wright–Giemsa-stained MLL-AF9–, MLL-AF9 E531R–, and MLL-AF9 D546R–expressing mouse BM cells following the third plating in methylcellulose (scale bar, 10 μm). F, Summary of immunofluorescence staining and one representative flow cytometry plot of MLL-AF9 and MLL-AF9 E531R cells from E for hematopoietic stem and differentiation surface markers. Relative mean fluorescence intensity (MFI) of staining is shown. Data are from five independent experiments; **, P < 0.01. G, Survival curves for sublethally irradiated mouse recipients (n = 10 for each cohort) of BM cells transformed with MLL-AF9 WT (blue) or MLL-AF9 E531R (red). Statistical significance was determined using a log-rank (Mantel–Cox) test. For details, see also Supplementary Fig. S3.
- Figure 5.
The BCOR/MLL-AF9 interaction regulates a unique set of genes. A, Left, volcano plot showing differential RNA expression in MLL-AF9 E531R samples compared with WT. Genes with FDR < 0.05 and altered >2-fold are shown in orange. Those with FDR < 0.05 and altered <2-fold are shown in red. Genes selected for qPCR are shown in blue. Ccnd1–3 are indicated in green. Right, MLL-AF9 E531R–altered genes that are direct MLL-AF9 targets. Log2 (fold change) is reported relative to WT. Genes labeled in C are highlighted in blue. B, Heatmap of genes with >2-fold expression change between MLL-AF9 WT and MLL-AF9 (E531R), with increased expression in red and decreased in blue. C, Venn diagram showing overlap of genes altered >2-fold by MLL-AF9 E531R (pink) with MLL-AF9 ChIP targets (blue; ref. 30) and genes showing loss of H3K79me2 (yellow) or H3K79me3 (green) in MLL-AF9 D546R samples (5). D, GSEA analysis identified “HALLMARK_MYC_TARETS_V1” signature based on comparison of MLL-AF9 and MLL-AF9 (E531R) RNA-seq data. E, Western blot analysis for Myc protein in MLL-AF9– and MLL–AF9 (E531R)–expressing cells. Myc protein level was quantified relative to actin from three independent experiments using the Fiji distribution of ImageJ.
- Figure 6.
Comparison of MLL-AF9 E531R (disruption of BCOR binding) RNA-seq data to RNA-seq data from Stavropolou and colleagues (35), identifying genes with increased expression in MLL-AF9 HSCs versus MLL-AF9 GMPs. Top, Venn diagram showing overlap of genes altered in the MLL-AF9 E531R gene set and increased in MLL-AF9–expressing HSCs versus MLL-AF9–expressing GMPs. The table lists all the genes in the overlap set that are increased in MLL-AF9 HSCs versus GMPs and that are down in the MLL-AF9 E531R gene set. The genes in the table that have been shown by ChIP-seq to be either SIX1 and SIX2 targets or solely SIX2 targets are colored in red and green, respectively. Bottom, Venn diagram showing overlap of genes altered in the MLL-AF9 E531R gene set and increased in MLL-AF9–expressing GMPs versus MLL-AF9–expressing HSCs. The table lists all the genes in the overlap set that are increased in MLL-AF9 GMPs versus HSCs and increased in the MLL-AF9 E531R gene set. The genes in the table are colored as above.
- Figure 7.
EYA1/SIX1 partially rescues phenotype from abrogated MLL-AF9/BCOR binding and schematic of BCOR/MLL-AF9–dependent gene regulation. A, Survival curves for sublethally irradiated mouse recipients (n = 5–10 per cohort) of BM cells transformed with MLL-AF9 WT (blue), MLL-AF9 E531R (1×; red), MLL-AF9 E531R (NC; red, cross), MLL-AF9 E531R (10×; maroon), MLL-AF9 E531R + Eya1 (green, triangle), or Eya1 alone (green, open diamond). All mice received the same number of transduced cells except those indicated (10×) which received 10-fold more cells. All mice received cells that had been cultured one week in methylcellulose prior to transplant except those indicated as (NC) that had no in vitro culture prior to transplant. Statistical significance compared with MLL-AF9 E531R was determined using a log-rank (Mantel–Cox) test. B, Images of Wright–Giemsa-stained mouse BM cells expressing MLL-AF9, MLL-AF9 E531R, and MLL-AF9 E531R coexpressing Eya1, SIX1, or both Eya1 and SIX1, following the third plating in methylcellulose (scale bar, 10 μm). C, Summary of immunofluorescence staining of MLL-AF9 E531R cells from B for relevant hematopoietic stem and differentiation surface markers. Relative mean fluorescence intensity (MFI) of staining is shown. Data are from three independent experiments; *, P < 0.05. D, Schematic of key effects of loss of BCOR/MLL-AF9 interaction. The levels of expression of Eya1, Six1, Hoxa7, and Cyclins D1–3 are regulated by the interaction of BCOR with WT MLL-AF9. In the absence of the MLL-AF9/BCOR interaction, levels of Eya1 and Six1 are decreased, as is the level of Myc protein, reducing the LIC population and resulting in a lack of in vivo leukemogenesis. The levels of Hoxa7 and Cyclins D1–3 are increased, resulting in increased proliferation as seen in the colony-forming assay.
Additional Files
Supplementary Data
- Supplementary Data - Supplementary figures, tables, and methods.
Volume 1, Issue 2
