Enhancer control of V(D)Jrecombination at the TCRβ locus: differential effects on DNA cleavage and joining

Abstract

Deletion of the TCRβ transcriptional enhancer (Eβ) results in nearly complete inhibition of V(D)J recombination at the TCRβ locus and a block in αβ T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in Eβ-deleted (Eβ−/−) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking Dβ andJβ gene segments in Eβ−/−thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRβ locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both Eβ−/− and Eβ+/+ thymocyte nuclei. However, the corresponding Dβ-to-Jβ coding joints are further reduced (by 100- to 300-fold) in Eβ−/−thymuses. Formation of extrachromosomal Dβ-to-Jβ signal joints appears to be intermediately affected and nonstandardDβ-to-Dβ joining occurs at the Eβ-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRβ recombination in the absence of the Eβ element and suggest an additional function for Eβ in the process of DNA repair at specific TCRβ sites during the late phase of the recombination reaction.