Three-way contact analysis characterizes the higher-order organization of the Tcra locus

The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Ea has been implicated in regulating the accessibility of Va and Ja genes through long-range interactions during T cell antigen receptor gene Tcra rearrangements. However, direct evidence for Ea physically mediating the interaction of Va and Ja genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher-order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multi-way contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Ja genes, Va genes, and Ea in CD4+CD8+ double-positive thymocytes. Notably, the INT2-TEAp loop emerged as a prominent structure likely responsible for bringing the proximal Ja genes and the Va genes into proximity. Moreover, the enhancer Ea utilizes this loop to establish physical proximity with the proximal Va gene region. This study provides insights into the higher-order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination. Overall design: Examination of 3C-HTGTS or 4C-seq NGS data involving pair-wise or/and three-way chromatin interactions from different viewpoints respectively before or/and after EACBE knock out (duplicates).