Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor ␣2 (CBF␣2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10-to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBF␣2. Furthermore, two additional lines of evidence indicated that CBF␣2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors.Auto-inhibition modulates a variety of transcription factor activities (21). In this regulatory mechanism, inhibitory sequences act in cis to repress such functions as DNA binding, transcriptional activation, nuclear localization, and ligand interaction. The picture emerging from the study of several transcription factors suggests that protein partnerships can counteract auto-inhibition. For example, serum response factor counteracts the auto-inhibition of Elk-1 DNA binding (36). The DNA binding of Pip, which contains an auto-inhibitory domain, requires interactions with PU.1 (9), and Pbx activates the DNA binding of its partner protein Hoxb (10, 11). Similarly, DNA binding by the yeast repressor a1 requires formation of a ternary complex with ␣2 (63). Examples within basal transcriptional machinery include the auto-inhibition of the subunit of Escherichia coli RNA polymerase. Interaction with core RNA polymerase counters this inhibitory effect (13). Also, the inhibitory function of the amino terminus of TATAbinding protein is abrogated by interactions with SNAPc (40). In each of these DNA binding scenarios, auto-inhibition generates a tighter control switch from the off to on state. To decipher the molecular basis of the interplay between autoinhibition and protein partnerships, we focus on Ets-1, for which auto-inhibition is well characterized at the mechanistic and structural levels (21).Auto-inhibition, together with protein partnerships, c...