Mechanism of c-Myb–C/EBPβ Cooperation from Separated Sites on a Promoter

c-Myb, but not avian myeloblastosis virus (AMV) v-Myb, cooperates with C/EBPβ to regulate transcription of myeloid-specific genes. To assess the structural basis for that difference, we determined the crystal structures of complexes comprised of the c-Myb or AMV v-Myb DNA-binding domain (DBD), the C/EBPβ DBD, and a promoter DNA fragment. Within the c-Myb complex, a DNA-bound C/EBPβ interacts with R2 of c-Myb bound to a different DNA fragment; point mutations in v-Myb R2 eliminate such interaction within the v-Myb complex. GST pull-down assays, luciferase trans-activation assays, and atomic force microscopy confirmed that the interaction of c-Myb and C/EBPβ observed in crystal mimics their long range interaction on the promoter, which is accompanied by intervening DNA looping.