DISTINCT ROLES FOR P-CREB AND LEF-1 IN TCR-ALPHA ENHANCER ASSEMBLY AND ACTIVATION ON CHROMATIN TEMPLATES IN-VITRO

The distal enhancer of the T-cell receptor (TCR) ex chain gene has bec ome a paradigm for studies of the assembly and activity of architectur al enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cel l transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-re sponsive transcription factor CREB. When added in combination, these f actors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell protei ns (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolis hed by a point mutation affecting the CREB PKA phosphorylation site (S er-133). Interestingly, LEF-1 strongly enhanced the binding of the AML 1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain was sufficient to f orm a higher-order complex with AML1/Ets-1, but exhibited only partial activity in transcription. We conclude that the T cell-enriched prote ins assemble on the enhancer independently of CREB and function synerg istically with CREB to activate the TCR alpha enhancer in a chromatin environment.