Hernandez-Munain, C.; Rodriguez-Caparros, A.; Sune, C.; Duarte-Ruiz, M.; Jimenez-Lozano, S.; Terron-Camero, L. C.; Lopez-Castellanos, L.; Andres-Leon, E.; Castro-Ruiz, V.; Lopez-Ros, J.; Moreno-Castillo, A.

Long-range functional enhancer-promoter (E-P) interactions typically occur within topologically associating domains (TADs), which facilitate contacts while restricting inter-domain communication. Although approximately one-third of E-P interactions cross TAD boundaries, they are considered non-functional and are only rarely reported during embryonic development. Here, we investigated the activity of the strong Tcra enhancer (Ea) positioned at the boundary between a centromeric TAD containing the T-lineage-specific Tcra-Tcrd locus and a telomeric TAD encompassing broadly expressed Dad1-to-Cdh24 genes. To directly assess Ea activity across TADs, we generated mice lacking Ea while preserving its associated CTCF-binding elements. As expected, Ea was required for T-cell specific Tcra-Tcrd transcription and normal T-cell development. In contrast, Ea did not activate Dad1 or Haus4 transcription in the telomeric TAD, indicating effective insulation by boundary elements. Unexpectedly, Ea selectively activated developmentally regulated Cdh24 expression, the most distal gene in the telomeric TAD, in thymocytes. These findings reveal a naturally occurring, functional cross-TAD E-P interaction during adult T-cell development, demonstrating that enhancers positioned at TAD boundaries can bypass topological insulation to selectively regulate distal gene expression.