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

Abstract Enhancer-promoter interactions are largely constrained within topologically associating domains (TADs), thereby contributing to regulatory insulation between adjacent domains. Although chromatin contacts frequently span TAD boundaries, their functional significance remains poorly understood, and functional enhancer-dependent regulation across TAD boundaries has been documented in only a limited number of developmental contexts. Here, we investigate the activity of the Tcra enhancer (Eα), located at the boundary between a centromeric TAD containing the T-lineage-specific Tcra-Tcrd locus and a telomeric TAD encompassing the Dad1-Acin1 gene cluster. By deleting Eα while preserving its adjacent CTCF-binding elements (EαCBEs), we dissected the regulatory contribution of Eα from that of the boundary-associated CTCF sites. Loss of Eα impaired Tcra-Tcrd expression and αβ T-cell development, whereas expression of most genes within the neighboring telomeric TAD remained largely unaffected. Unexpectedly, Eα was required for normal Ajuba and Cdh24 expression during thymocyte development, two distal genes in the neighboring TAD located more than 350 and 400 kb away from the enhancer, respectively. Thus, Eα regulates a restricted subset of distal genes across a TAD boundary while leaving more proximal genes largely unaffected. Notably, Acin1 expression was also unaffected despite strong interactions between Acin1 and Eα+EαCBE regions. These findings reveal that long-range chromatin interactions do not necessarily predict functional enhancer responsiveness and demonstrate that an enhancer positioned at a TAD boundary can exert highly selective regulatory effects within a neighboring domain.