Locus-specific spatial and transcriptional dynamics direct T cell lineage choice in the thymus

Decades of investigation have made the choice between CD4 and CD8 T cell lineages in the thymus a key paradigm for mammalian cell commitment and differentiation, yet how transcriptional diversification is coordinated with 3D genome organisation remains largely unexplored. Here, using single-molecule transcript quantification and region capture Pico-C (RCPC), we resolve distinct transcriptional and spatial chromatin dynamics at the lineage-defining loci Cd4 and Cd8. We find early Cd4/Cd8 transcriptional divergence driven by MHC-dependent bias in Cd8a transcript copy number, which unfolds gradually across both CCR7− and CCR7+ differentiation stages. Unexpectedly, Cd4 and Cd8 loci show fundamentally different spatial contact dynamics: transiently reduced mesoscale (10-100kb) contacts at Cd8 provide a permissive chromatin environment throughout the transcriptional diversification process. By contrast, Cd4 undergoes switch-like contact changes limited to the microscale (1kb) late in differentiation, associated with lineage stabilization by ThPOK and/or RUNX3. These results place distinct, locus-specific chromatin environments at the centre of transcriptional diversification during T cell lineage choice.