TCRalpha enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex

The TCR alpha enhancer (Ealpha) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ealpha occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ealpha occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Ealpha is known to be active (CD4(+)CD8(+)-DP cells), but surprisingly, also in cells at an earlier developmental stage where Ealpha is not active (CD4(-)CD8(-)-DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo-protein complex topography. Stage-specific differences in factor composition at Ealpha sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre-assembled nucleo-protein complex correlate with the onset of Ealpha activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.