Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development

T cell antigen receptor delta (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing gamma delta thymocytes. Here, we use linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4(-)CD8(-)double- negative thymocyte progenitors differentiated in vitro from bone marrow-derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement. We also find frequent, previously unappreciated, Trdd1 and Trdd2 rearrangements that inactivate Tcrd, including sequential rearrangements from V(D) J recombination signal sequence fusions. Moreover, we find dozens of RAG off-target sequences that are generated via RAG tracking both upstream and downstream from the Trdd2 recombination center across the Tcrd loop domain that is bounded by the upstream INT1-2 and downstream TEA elements. Disruption of the upstream INT1-2 boundary of this loop domain allows spreading of RAG on-and off-target activity to the proximal Trdv domain and, correspondingly, shifts the Tcrd V(D) J recombination landscape by leading to predominant V(D) J joining to a proximal Trdv3 pseudogene that lies just upstream of the normal boundary.