DNA binding and oligomeric assembly of the RUNX1-β/SMMHC complex. COS7 cells were transfected with each of the indicated expression plasmid (2 μg), and WCE was prepared. (A) A fixed amount of RUNX1 and WCE containing equivalent amount of either PEBP2β1 or β/SMMHC together with wild-type DNA probe were mixed as indicated and incubated, and EMSA was performed. Competitor oligonucleotides with wild-type (WT) or mutated RUNX-binding sites (MT) were incubated in the reaction mixture at 5-, 25-, and 100-fold molar excess. Migration of RUNX1 or RUNX1/β heterodimer is shown in lanes 1 and 9, respectively. Note that the complex formed by β/SMMHC, RUNX1, and DNA did not migrate into the gels but was eliminated specifically by WT (lanes 16-24). (B) A fixed amount of RUNX1 was mixed with an equivalent amount of the indicated protein and subjected to EMSA. (C) Assessment of the oligomeric state of β/SMMHC by glutaraldehyde cross-linking. Proteins overexpressed in COS7 cells were cross-linked in vitro with glutaraldehyde and analyzed by 4% to 20% gradient SDS-PAGE followed by immunoblotting with monoclonal anti-Cbfβ, as described in “Materials and methods.” Symbols on the right panel indicate the oligomeric state of cross-linked products: arrowhead indicates tetramer; and arrow, dimer. (D) Dominant binding of β/SMMHC over PEBP2β depends on the hyperdimerization domain. A fixed amount of RUNX1 (1 unit) and increasing relative amounts (0.25, 0.5, and 1 unit) of PEBP2β or β/SMMHC or its derivatives were subjected to EMSA (lanes 1-21). Similar experiments were carried out with increasing amounts of β/SMMHC constructs and a fixed amount of PEBP2β1 (1 unit; lanes 22-36). Note that A-ΔE33-36 has largely lost the ability to act dominantly over PEBP2β1.