Fig. 2.

Specific binding of GFP-M&M to MYB-binding sites, recruitment of AML1-ETO, and repression of MYB-responsive promoters by AML1-ETO in the presence of GFP-M&M. (A) In vitro recruitment of AML1-ETO to MYB-binding sites. Nuclear extracts from Cos cells transfected with Myb, GFP-M&M, and AML1-ETO as indicated were analyzed by an electrophoretic mobility-shift assay (EMSA). Competition experiments with specific MYB and nonspecific oligonucleotides demonstrated the specificity of M&M binding. The supershift of M&M cotransfected with AML1-ETO evidenced the recruitment of AML1-ETO to MYB-binding sites. (B) Recruitment of AML1-ETO in vivo. KCL22 cells transfected with FLAG-AML1-ETO and GFP or GFP-M&M were crosslinked with formaldehyde, lysed, sonicated, and immunoprecipitated with anti-Flag or with unspecific mouse IgG. Immunoprecipitated chromatin was analyzed by PCR for the KIT promoter region and the p14^ARF promoter region. One representative of two experiments with similar results is shown. (C) Expression of FLAG-AML1-ETO and GFP/GFP-M&M in the transfected cells. The expression level of FLAG-AML1-ETO and GFP/GFP-M&M in transfected cells was detected by Western blot. (D) KCL22 cells were transiently transfected with a MYB-responsive luciferase construct and AML1, AML1-ETO, GFP-ΔM&M, and GFP-M&M (as indicated). Firefly luciferase values were standardized to expression of a cotransfected Renilla luciferase vector construct. Results are shown as means and SEM of three independent experiments. (E) Primary murine bone marrow cells were transduced with GFP or GFP-M&M and analyzed for the expression of KIT. The results of one of two independent experiments are shown.