American Society of Hematology

Figure 3

$Figure 3. Functional analysis of RUNX1 mutants in vitro. (A) Transcriptional potential of the RUNX1 mutants in 293T cells. Cells were cotransfected with 0.5 μg of pM-CSF-R-luc reporter plasmid, 0.5 μg of flag-CBFβ expression plasmid, the indicated amount of RUNX1 expression plasmid, and 0.05 μg of pRL-SV40 as an internal control. The myc-WT RUNX1–encoding plasmid (0.5 μg) was cotransfected with increasing doses (0.5, 1, and 1.5 μg) of expression vectors containing the indicated RUNX1 mutants. Each value represents the mean of 3 independent experiments. The relative luciferase units are expressed as average ± SD. (B) DNA-binding potential of RUNX1 mutants analyzed by gel-shift assay using nuclear extracts from 293T cells transfected with WT or mutated RUNX1 expression plasmids. (C) ChIP-qPCR assay of the IL-3, GM-CSF, M-CSFR, MPO, and TCRβ promoters in 32D cells. WT RUNX1 and G381fs-ter570 proteins, but not H78Q, V91fs-ter94, R139G, D171G, R174Q, or R293X mutant proteins, were enriched on these promoters. (D) Heterodimerization ability of RUNX1 mutants with CBFβ. 293T cells were transfected transiently with flag-CBFβ together with myc-WT RUNX1 or mutants. Upper panel shows that the immunoprecipitation of whole-cell lysates by anti-flag Abs coprecipitates the indicated myc-RUNX1 proteins; bottom panel shows the corresponding CBFβ expression levels analyzed by anti-CBFβ Ab. The numbers (1-7) represent various RUNX1 mutants that are also shown in panel C and in Figure 2A. The 2 different gels are indicated by the gray dividing lines. (E) RUNX1 mutants compete with WT to bind CBFβ. Ectopic expression of RUNX1 mutants could impair the interaction between WT RUNX1 and CBFβ. 293T cells were transfected with flag-CBFβ and myc-tagged WT RUNX1 or with RUNX1 mutants. After 48 hours, the proteins were prepared for anti-flag immunoprecipitation, followed by WB with anti-flag and anti-myc Abs. H78Q, R139G, D171G, R174Q, and G381fs-ter570, especially R293X, but not V91fs-ter94, could compete with WT to bind CBFβ. (F) Interaction between WT and mutated RUNX1 proteins. 293T cells were transiently transfected with flag-RUNX1 together with myc-WT RUNX1 or mutants. After 48 hours, whole-cell lysates were immunoprecipitated with anti-flag Ab and analyzed by WB with anti-myc (top panel) and anti-flag (bottom panel) Abs. Flag-RUNX1 efficiently interacts with myc-WT RUNX1, H78Q, R139G, D171G, and R174Q, but not with V91fs-ter94, R293X, or G381fs-ter570. (G) Gel-filtration analysis. Protein fractions collected from the indicated elution volumes were analyzed by WB using anti-myc Ab. Vertical lines show the positions of the corresponding size standards.$

Functional analysis of RUNX1 mutants in vitro. (A) Transcriptional potential of the RUNX1 mutants in 293T cells. Cells were cotransfected with 0.5 μg of pM-CSF-R-luc reporter plasmid, 0.5 μg of flag-CBFβ expression plasmid, the indicated amount of RUNX1 expression plasmid, and 0.05 μg of pRL-SV40 as an internal control. The myc-WT RUNX1–encoding plasmid (0.5 μg) was cotransfected with increasing doses (0.5, 1, and 1.5 μg) of expression vectors containing the indicated RUNX1 mutants. Each value represents the mean of 3 independent experiments. The relative luciferase units are expressed as average ± SD. (B) DNA-binding potential of RUNX1 mutants analyzed by gel-shift assay using nuclear extracts from 293T cells transfected with WT or mutated RUNX1 expression plasmids. (C) ChIP-qPCR assay of the IL-3, GM-CSF, M-CSFR, MPO, and TCRβ promoters in 32D cells. WT RUNX1 and G381fs-ter570 proteins, but not H78Q, V91fs-ter94, R139G, D171G, R174Q, or R293X mutant proteins, were enriched on these promoters. (D) Heterodimerization ability of RUNX1 mutants with CBFβ. 293T cells were transfected transiently with flag-CBFβ together with myc-WT RUNX1 or mutants. Upper panel shows that the immunoprecipitation of whole-cell lysates by anti-flag Abs coprecipitates the indicated myc-RUNX1 proteins; bottom panel shows the corresponding CBFβ expression levels analyzed by anti-CBFβ Ab. The numbers (1-7) represent various RUNX1 mutants that are also shown in panel C and in Figure 2A. The 2 different gels are indicated by the gray dividing lines. (E) RUNX1 mutants compete with WT to bind CBFβ. Ectopic expression of RUNX1 mutants could impair the interaction between WT RUNX1 and CBFβ. 293T cells were transfected with flag-CBFβ and myc-tagged WT RUNX1 or with RUNX1 mutants. After 48 hours, the proteins were prepared for anti-flag immunoprecipitation, followed by WB with anti-flag and anti-myc Abs. H78Q, R139G, D171G, R174Q, and G381fs-ter570, especially R293X, but not V91fs-ter94, could compete with WT to bind CBFβ. (F) Interaction between WT and mutated RUNX1 proteins. 293T cells were transiently transfected with flag-RUNX1 together with myc-WT RUNX1 or mutants. After 48 hours, whole-cell lysates were immunoprecipitated with anti-flag Ab and analyzed by WB with anti-myc (top panel) and anti-flag (bottom panel) Abs. Flag-RUNX1 efficiently interacts with myc-WT RUNX1, H78Q, R139G, D171G, and R174Q, but not with V91fs-ter94, R293X, or G381fs-ter570. (G) Gel-filtration analysis. Protein fractions collected from the indicated elution volumes were analyzed by WB using anti-myc Ab. Vertical lines show the positions of the corresponding size standards.

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