Loop Stacking Attenuates Chromatin Boundaries in TCR Loci During Thymocyte Development

Topologically Associating Domains (TADs) partition the genome into self-interacting regions through boundaries. Chromatin configuration plays a crucial role in TCR gene rearrangements, thereby affecting repertoire diversity. To assess the dynamics of chromatin boundaries within TCR genes, we conducted Hi-C assays on double-negative (DN) and double-positive (DP) cells from RAG-deficient mice. A chromatin boundary at the 3' end of the Tcrb locus observed in DN cells dissipated in DP cells, thereby altering interactions between the main Vß cluster and the recombination center. Additionally, the INTs boundary in the Tcra-Tcrd locus disappeared in DP cells, leading to enhanced interactions between the proximal Va region and recombination center at the Ja segments. Subsequent analysis revealed that the disappearance of boundaries was not attributable to diminished CTCF binding but rather heightened activity within these regions. This study illuminates the developmental dynamics of TAD boundaries in TCR genes, enriching our understanding of their evolving nature. Overall design: Hi-C analysis of EACBE+/+Rag1-/- and EACBE-/-Rag1-/- DN thymocytes;Hi-C analysis of EACBE+/+Rag1-/- DP thymocytes;3C-HTGTS analysis of EACBE+/+Rag1-/- DN and/or DP thymocytesusing different baites(Trbv12-2,Trbv5,Eb,Trav21,Trdv2-2);3C-HTGTS analysis of EACBE+/+Rag1-/- and/or EACBE-/-Rag1-/- DN thymocytes using different baites(Ea,INT2,Trdv2-2,Trdd2,Trdv5,TEA);HTGTS-VDJ analysis of EACBE+/+Rag1-/- and EACBE-/-Rag1-/- DN or DP thymocytes at the baits from Trdj1 or Traj61;ATAC-seq analysis of EACBE+/+Rag1-/- and EACBE-/-Rag1-/- DN thymocytes;ATAC-seq analysis of EACBE+/+Rag1-/- DP thymocytes.