COOPERATIVE BINDING OF ETS-1 AND CORE BINDING-FACTOR TO DNA

Two phorbol ester-inducible elements (betaE2 and betaE3) within the hu man T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor f amilies. Recombinant Ets-1 and purified CBF bound individually to beta E2 and betaE3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to betaE2 and betaE3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to betaE2. Formation of Ets-1-CBF-DNA complexe s increased the affinity of Ets-1-DNA interactions and decreased the r ate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial req uirements for the cooperative interaction of Ets-1 and CBF were analyz ed by oligonucleotide mutagenesis and binding site selection experimen ts. Core and Ets sites were coselected, and there appeared to be littl e constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA -binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant . Ets and core sites are found in several T-cell-specific enhancers, s uggesting that this interaction is of general importance in T-cell-spe cific transcription.