(A) Diagrammatic representation of CBFβ-SMMHC fusion variants. CBFβ-SMMHC_d134-236, generated by type I fusion, misses the HABD as well as CBFβ residues encoded by exon 5. CBFβ-SMMHC_d179-221 misses only the HABD and has been described before (Lukasik et al., 2002).
(B) ITC measurement of interactions between 200 μM CBFβ-SMMHC_d134-236 and 7.5 μl injections of 15 μM RUNX1 Runt domain. The top panel shows the raw data while the bottom panel is a plot of the binding corrected for the dilution enthalpy (average dilution enthalpy = 232 cal mol⁻¹). Data were fit to a one-site binding model. The results of a fit to one titration are shown in the box at the lower right corner. The average K_d of two independent experiments is 709 (± 47) nM.
(C) CD4 reporter assay. E4P4: CD4 enhancer and promoter; P131-265: core sequence of CD4 repressor; FL: CBFβ-SMMHC; d179-221: CBFβ-SMMHC_d179-221; d134-236: CBFβ-SMMHC_d134-236. Δ: statistically significant difference (P<0.05) between the top two conditions, which are P131-265 + Runx1 with either d179-221 or d134-236. *: statistically significant differences (P<0.05) between the top two conditions and the third one, which is P131-265 + Runx1 + FL.
(D) MCSFR reporter assay. pMCSFR: the luciferase reporter driven by human MCSFR promoter.
(E and F) Western blot analysis showing the expression of the transfected constructs in the reporter assays (C and D, respectively). The expression of CBFβ and full-length and variant CBFβ-SMMHC constructs was detected with a mouse monoclonal antibody (β141.2) specific for CBFβ.
The error bars in (C) and (D) represent one standard deviation.

(A) Targeting strategy used to replace exon5 of Cbfb with the targeting constructs. Location of probe 0.2C and the sizes of NcoI fragments detected by 0.2C are indicated. The arrows indicate the locations of PCR primers used for genotyping. Filled triangles represent lox-P sites.
(B) Southern blot hybridization of NcoI-digested DNA from the parental ES cells (TC1; lanes 1, 3, and 5) and the targeted ES cell clones (lane 2: clone #10 for Cbfb-MYH11; lanes 4 and 6: clones #220 and #269 for Cbfb-MYH11_d179-221) with probe 0.2C. The 15.7 kb band corresponds to the wild-type Cbfb allele and the 6.8 kb band corresponds to the knock-in allele.
(C) Western blot analysis of ES cells. Lane 1: the parental ES cell line TC-1; Lane 2: ES cell line #10 (knock-in of full-length Cbfb-MYH11), Lanes 3 and 4: ES cell lines #220 and #269 with knock-in of Cbfb-MYH11_d179-221. The antibody used was the mouse monoclonal antibody (β141.2) specific for CBFβ. The calculated molecular weights for CBFβ-SMHHC, CBFβ-SMHHC_d179-221, and CBFβ are 71 KD, 65 KD, and 22 KD, respectively.

(A) Genotype and phenotype of embryos derived from crosses between Cbfb–MYH11 chimeras and normal females. +/+ are wild type littermates, +/d179-221 are embryos heterozygous for Cbfb-MYH11_d179-221 knock-in. Viability was determined by embryo heart beats.
(B–D) Histologic sections of fetal livers from E12.5 embryos. (B) wildtype control, (C) Cbfb^{+/MYH11d179-221}, (D) Cbfb^+/MYH11. Sections were stained with H and E. Yellow arrows indicate hematopoietic cells in the livers. Blue arrows indicate megakaryocytes. Scale bars represent 100 μm.
(E–H) In vitro differentiation assay of fetal liver hematopoietic cells. Panels (E) and (F) compare colony numbers from embryos of wildtype (black bars) and Cbfb^+/MYH11_d179-221 (gray bars). Panels (G) and (H) compare colony numbers from embryos of wildtype (black bars) and Cbfb^+/MYH11 (gray bars). Panels (E) and (G): total colony numbers. BFU-E: burst-forming unit-erythroid; CFU-E: colony-forming unit-erythroid; CFU-G: colony-forming unit-granulocyte; CFU-GM: colony-forming unit-granulocyte/macrophage; CFU-GEMM: colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte; CFU-M: colony-forming unit-macrophage. The error bars represent one standard deviation. **: P<0.001

(A) Survival curves of Cbfb^+/MYH11_d179-221 and Cbfb^+/MYH11 chimeras. Dotted line: the survival curve of Cbfb^+/MYH11 chimeras (n=17); solid line: the survival curve of Cbfb^+/MYH11_d179-221 chimeras (n=22). The mice were not treated with any mutagens such as ENU.
(B) Morphology of leukemic cells in the Cbfb^+/MYH11_d179-221 mice. Left panel: wright-Giemsa stained peripheral blood smears showing poorly differentiated stem cell like cells (arrowheads) and myeloblasts (arrows). Middle and right panels: H&E stained bone marrow sections from mice of the indicated genotypes. Scale bar in the left panel represent 10 μm; those in the middle and right panels represent 50 μm.
(C) FACS analysis of peripheral blood cells in a Cbfb^+/MYH11_d179-221 mouse using antibodies against c-kit, Mac-1, GR-1, CD131, B220, CD4 and CD8.
(D) Correlation between cell morphology and surface marker expression for leukemic cells from the Cbfb^+/MYH11_d179-221 mice. FACS sorted c-kit⁺/GR1⁻/Mac1⁻ and c-kit⁻/GR1⁺/Mac1⁺ populations from mice of the indicated genotypes were analyzed for morphologic features through Wright-Giemsa staining of cytospin preparations. Scale bars represent 10 μm.
(E) The percentages of leukemia cells expressing c-kit or Gr-1/Mac1 in the Cbfb^+/MYH11 and Cbfb^+/MYH11_d179-221 mice (N = 5 for each genotype) as detected by FACS. The error bars represent one standard deviation.
(F and G) Expression of the knocked-in fusion genes in the leukemia cells. (F) RT-PCR with RNA samples from ES cells (lanes 1–4) and leukemic cells (lanes 5–7) with PCR primers flanking the Cbfb and MYH11 fusion junctions in Cbfb-MYH11 and Cbfb-MYH11_d179-221. Lane 1: ES cell line TC1; Lane 2: ES cell clone #10 with knock-in of Cbfb-MYH11; Lanes 3 and 4: ES cell lines #220 and 269 with knock-in of Cbfb-MYH11_d179-221; Lane 5: leukemia cells from a Cbfb^+/MYH11 chimera; Lanes 6 and 7: leukemia cells from two Cbfb^+/MYH11_d179-221 chimeras. (G) Western blot analysis with protein samples from leukemic cells of the indicated genotype and the CBFβ-specific antibody (β141.2).

(A) and (B) In vitro differentiation of human CD34+ cells. Human CD34+ cells were transduced with empty retroviral vector (MIG), and vectors expressing CBFB-MYH11 (FL), CBFB-MYH11_d179-221 (d179-221), or CBFB-MYH11_d134-236 (d134-236). The transduced cells were sorted by FACS and two thousand GFP⁺ cells were plated in serum-free methylcellulose cultures. (A) Total colony numbers scored after 14 days. The total colony number from MIG-transduced cells was set at 1, and the total colony numbers from the other two transduced cell populations were calculated relative to that of MIG. The data shown are averages of three independent experiments. (B) Frequencies of BFU-E, CFU-GM, and CFU-GEMM colonies from transduced human CD34⁺ cells. The data shown are averages of two independent experiments. *: p<0.01 between clones transduced with MIG and those with CBFB-MYH11 constructs; **: p<0.001 between clones transduced with MIG and those with CBFB-MYH11 constructs. The error bars represent one standard deviation.
(C) Cell morphology from long-term cultures of human CD34+ cells transduced with CBFB-MYH11 or CBFB-MYH11_d179-221. Shown are cytospin preparations of non-adherent cells at week 10, stained with Wright-Giemsa stain. Scale bars represent 10 μm.
(D) Western blot analysis with protein samples from transduced human CD34+ cells and the CBFβ-specific antibody (β141.2).

(A) qRT-PCR analysis of RNA samples from human CD34+ cell transduced with MIG, CBFB-MYH11, and CBFB-MYH11_d179-221. Error bars represent SDs of 2 to 6 samples. *: P<0.05 between MIG and CBFB-MYH11 or CBFB-MYH11_d179-221; **: P<0.01 between MIG and CBFB-MYH11 or CBFB-MYH11_d179-221; ▲: P<0.05 between CBFB-MYH11 and CBFB-MYH11_d179-221. The error bars represent one standard deviation.
(B) Western blot detection of MN1 expression in leukemia cells from Cbfb^+/MYH11 and Cbfb^+/MYH11_d179-221 mice.
(C) Overexpression of myeloid genes in the Cebpa network from analysis of microarray data by Ingenuity Pathway Analysis (IPA). The blue arrows highlights connections directly from Cebpa. Pink colored genes are those that upregulated. More detailed information about the genes in this network is available in Table S1.
(D) The MN1 network identified by IPA analysis of microarray data. More detailed information about the genes in this network is available in Table S2.

(A) RUNX1 phosphorylation by CBFβ and deletion variants of CBFβ-SMMHC. CBFβ, CBFβ-SMMHC, or deletion variants of CBFβ-SMMHC fusion proteins were co-expressed in 293T cells with RUNX1 and wild-type p300. Western blot analyses were performed using the indicated antibodies.
(B) p300 phosphorylation by CBFβ and deletion variants of CBFβ-SMMHC fusion proteins. Wild-type p300 or mutant p300 (p300 ^ΔSTP1,2,3) were transfected with RUNX1 and CBFβ, CBFβ-SMMHC, or the CBFβ-SMMHC deletion constructs. Immunoblotting with p300 antibody was performed with lysates from transiently transfected 293T cells.
(C) RUNX1 phosphorylation in human and mouse leukemia samples. ME1: a cell line derived from an inv(16)+ AML case (Yanagisawa et al., 1991), primary: primary leukemia cells from bone marrow of an inv(16)+ AML case (Liu et al., 1996). Both human cases contain the type A CBFB-MYH11 fusion. FL: full length CBFβ-SMMHC, D43: CBFβ-SMMHC _d179-221, WT BM: wildtype whole bone marrow cells after erythrocyte lysis. Protein lysates were isolated from human and mouse leukemic cells and western blot analyses were performed using the indicated antibodies.

Working model for leukemogenesis in Cbfb^+/MYH11_d179-221 mice.