Selective cross-TAD gene regulation by the Tcra enhancer during T cell development

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 (E) 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 E activity across TADs, we generated mice lacking E while preserving its associated CTCF-binding elements. As expected, E was required for T-cell specific Tcra-Tcrd transcription and normal T-cell development. In contrast, E did not activate Dad1 or Haus4 transcription in the telomeric TAD, indicating effective insulation by boundary elements. Unexpectedly, E 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.