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 <i>Tcra</i> and <i>Tcrd</i> genes reside within a shared genomic locus, with <i>Tcrd</i> rearrangement occurring first in the double-negative (DN) stage during thymocyte development. Elucidating the regulatory mechanisms governing <i>Tcrd</i> rearrangement is therefore crucial for understanding the developmental coordination of both <i>Tcrd</i> and <i>Tcra</i> 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 <i>Tcra</i>-<i>Tcrd</i> locus, regulates <i>Tcrd</i> rearrangement. EACBE promotes the usage of proximal V_δ gene segments by facilitating spatial proximity between the <i>Tcrd</i> recombination centre and these V_δ elements. Notably, EACBE counteracts the insulating effects of INTs, two CBEs that demarcate the proximal V region from the D_δ-J_δ-C_δ cluster, thereby enabling effective chromatin extrusion. Furthermore, EACBE indirectly shapes the <i>Tcra</i> repertoire through its influence on <i>Tcrd</i> 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.