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_α-to-J_α 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_α) interacts directly with V_α and J_α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E_α promotes interactions between these V_α and J_α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E_α and to many locus promoters, biases E_α to interact with these promoters, and is required for efficient V_α–J_α recombination. Our data indicate that E_α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V_α–J_α synapsis and recombination.