title: Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.
abstract: T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ 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.
authors.affiliation: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: Lijuan
authors.identifier: 0000-0003-1288-3210
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: Richard L
authors.identifier: 0000-0001-6963-4931
authors.initials: RL
authors.lastname: Frock
authors.affiliation: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Du
authors.affiliation: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: Jiazhi
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC 27710 michael.krangel@duke.edu alt@enders.tch.harvard.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Krangel
authors.affiliation: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115 michael.krangel@duke.edu alt@enders.tch.harvard.edu.
authors.forename: Frederick W
authors.identifier: 0000-0002-0583-1271
authors.initials: FW
authors.lastname: Alt
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D018398:Homeodomain Proteins / Q000502:physiology*; D051379:Mice; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D013601:T-Lymphocytes / Q000502:physiology*; D060152:V(D)J Recombination
chemical_list: D026923:Amino Acid Transport Systems, Basic; D018398:Homeodomain Proteins; D016692:Receptors, Antigen, T-Cell, gamma-delta; C064658:RAG-1 protein; C087435:Slc7a2 protein, mouse