Flexible Stereospecific Interactions and Composition within Nucleoprotein Complexes Assembled on the TCRα Gene Enhancer

Abstract

During thymocyte maturation, enhancers of genes encoding for TCRδ (Tcrd) and TCRα (Tcra), Eδ8, and Eα, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Eα fragment, Tα1-Tα2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Eα or Tα1-Tα4. These experiments indicated that Tα3-Tα4 collaborates with factors bound to Tα1-Tα2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Tα1-Tα2 explained the molecular basis for requirement of intact Tα2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Eα, EαMC, in which Eδ myb and runx sites have been substituted for Tα2 runx and ets sites, that argues against the notion of a requirement for strict Eα enhanceosome structure for function. EαMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Eα enhanceosome are rather flexible. Activation of V(D)J recombination by EαMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Tα1-Tα2. These results indicate that Tα3-Tα4 itself is not sufficient to impart a developmental delay to a chimeric “early” enhancer, and indicate the need for functional collaboration between Tα2 runx/ets sites binding proteins and proteins bound to Tα3-Tα4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Eα might represent a more flexible form of information processing during thymocyte development.