An HMG I/Y-containing repressor complex and supercoiled DNA topology are critical for long-range enhancer-dependent transcription in vitro

The 3' enhancer of the T cell receptor alpha-chain (TCR alpha) gene directs the tissue- and stage-specific expression and V(D)J recombination of this gene locus. Using an in vitro system that reproduces TCR alpha enhancer activity efficiently, we show that long-range promoter-enhancer regulation requires a T cell-specific repressor complex and is sensitive to DNA topology. In this system, the enhancer functions to derepress the promoter on supercoiled, but not relaxed, templates. We find that the TCR alpha promoter is inactivated by a repressor complex that contains the architectural protein HMG I/Y. In the absence of this repressor complex, expression of the TCR alpha gene is completely independent of the 3' enhancer and DNA topology. The interaction of the T cell-restricted protein LEF-1 with the TCR alpha enhancer is required for promoter derepression. In this system, the TCR alpha enhancer increases the number of active promoters rather than the rate of transcription. Thus, long-range enhancers function in a distinct manner from promoters and provide the regulatory link between repressors, DNA topology, and gene activity.