Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4(-)CD8(-) double-negative thymocytes and Tcra gene segments in CD4(+)CD8(+) double-positive thymocytes. Initial V-alpha-to-J(alpha) recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E-alpha) interacts directly with V-alpha and J(alpha) gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E-alpha promotes interactions between these V-alpha and J(alpha) segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E-alpha and to many locus promoters, biases E-alpha to interact with these promoters, and is required for efficient V-alpha-J(alpha) recombination. Our data indicate that E-alpha and CTCF cooperate to create a developmentally regulated chromatin hub that supports V-alpha-J(alpha) synapsis and recombination.