AN ENHANCER LEF-1 TCF-1 SITE IS ESSENTIAL FOR INSERTION SITE-INDEPENDENT TRANSGENE EXPRESSION IN THYMUS/

Transcriptional activation of eukaryotic genes involves assembly of sp ecific multiprotein complexes on the promoters and enhancers of the ge nes. Recently, it has been proposed that the role of some of the prote ins in the complex may be architectural, involving DNA bending, orches tration of protein-protein interaction and modulation of nucleosome st ructure, This role has been proposed for the HMG proteins LEF-1 and TC F-1. We examined the role of a LEF-1/TCF-1 binding site in the human a denosine deaminase (ADA) thymic enhancer. Mutational analysis demonstr ated that a functional LEF-1/TCF-1 binding site is not required for en hancer-mediated transcriptional activation in transient transfection s tudies, but is essential for enhancer function in the in vivo chromati n context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroy ed the ability of the ADA enhancer/locus control region to specify hig h level, insertion site-independent transgene expression in thymus, DN ase I and DpnII accessibility experiments indicated dramatic changes i n the chromatin organization of the ADA enhancer in transgenic mice wi th a mutated LEF-1/TCF-1 site, This supports the hypothesis that facto rs binding the LEF1/TCF-1 site play an architectural role during the i n vivo activation of the ADA enhancer, possibly involving chromatin mo dification.