The insulator EACBE regulates V(D)J recombination of Tcrd gene by modulating chromatin organization.

T cell receptor (TCR) diversity, essential for the recognition of a wide array of antigens, is generated through V(D)J recombination. The and genes reside within a shared genomic locus, with rearrangement occurring first in the double-negative (DN) stage during thymocyte development. Elucidating the regulatory mechanisms governing rearrangement is therefore crucial for understanding the developmental coordination of both and rearrangements. Chromatin architecture, orchestrated by CTCF-cohesin complexes and their binding sites, plays a fundamental role in regulating V(D)J recombination of antigen receptor genes. In this study, we report that EACBE, a CTCF binding element (CBE) located downstream of the - locus, regulates rearrangement. EACBE promotes the usage of proximal V gene segments by facilitating spatial proximity between the recombination centre and these V elements. Notably, EACBE counteracts the insulating effects of INTs, two CBEs that demarcate the proximal V region from the D-J-C cluster, thereby enabling effective chromatin extrusion. Furthermore, EACBE indirectly shapes the repertoire through its influence on rearrangement. These findings reveal a novel regulatory axis involving special chromatin configuration and highlight distinct roles for specific CTCF binding sites in modulating antigen receptor gene assembly.