In this study we examine the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins. We find that reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in the enhancer. We show that Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 was found previously to bend the DNA helix in the center of the TCR alpha enhancer. We now show that the HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites.
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