Flexible stereospecific interactions and composition within nucleoprotein complexes assembled on the TCR alpha gene enhancer.

During thymocyte maturation, enhancers of genes encoding for TCR delta (Tcrd) and TCR alpha (Tcra), E delta(8), and E alpha, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an E alpha fragment, T alpha 1-T alpha 2, which constitutes a well-characterized compact nueleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire E alpha or T alpha 1-T alpha 4. These experiments indicated that T alpha 3-T alpha 4 collaborates with factors bound to T alpha 1-T alpha 2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on T alpha 1-T alpha 2 explained the molecular basis for requirement of intact T alpha 2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of E alpha, E alpha MC, in which E delta myb and runx sites have been substituted for T alpha 2 runx and ets sites, that argues against the notion of a requirement for strict E alpha enhanceosome structure for function. E alpha MC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an E alpha enhanceosome are rather flexible. Activation of V(D)J recombination by E alpha MC during thymocyte development resulted, however, to be premature and indistinguishable from that of T alpha 1-T alpha 4. These results indicate that T alpha 3-T alpha 4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between T alpha 2 runx/ets sites binding proteins and proteins bound to T alpha 3-T alpha 4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on E alpha might represent a more flexible form of information processing during thymocyte development. The Journal of Immunology, 2009, 183: 1871-1883.