TCRα enhancer activation occurs via a conformational change of a pre‐assembled nucleo‐protein complex

The TCR α enhancer (Eα) 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 Eα occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Eα occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Eα is known to be active (CD4 + CD8 + –DP cells), but surprisingly, also in cells at an earlier developmental stage where Eα 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 Eα 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 Eα activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.