Mechanisms that diversify an otherwise intrinsically processive Trav-Traj recombination program

Abstract

Abstract In DP thymocytes, the Tcra locus undergoes multiple rounds of V-J rearrangement in order to generate a functional TCRα chain. While Traj segment usage is strictly ordered from 5′ to 3′ as rearrangement proceeds, it is unclear to what degree Trav usage is also ordered. To test this, we assessed the Trav-Traj combinatorial repertoire in murine DP thymocytes using 5′ RACE followed by paired end sequencing. In 129Sv/J WT thymocytes, we found a clear bias toward proximal-proximal and distal-distal joins, but Trav-Traj pairings were highly diverse, even in primary V-J rearrangement. However, combinatorial diversity was highly constrained in HYα KI thymocytes, in which a fixed primary V-J rearrangement in the endogenous Tcra locus is the starting point for secondary rearrangements. Thus, secondary rearrangement is highly processive through the V and J arrays, but surprising diversity is introduced by primary rearrangement. Tcrd rearrangement precedes Tcra rearrangement in developing thymocytes. In INT1-2KO mice, CTCF site deletion biases Trdv usage toward the most proximal gene segments. These mice, like HYα KI, displayed a substantially constrained Tcra repertoire, with limited diversity of primary rearrangements. Thus, variable truncation of the Trav array by prior Tcrd rearrangement creates diversity in an otherwise highly processive Tcra rearrangement program by imparting combinatorial diversity to primary Tcra rearrangement. This diversification is functionally important, as Trav1-Traj33 pairing and MAIT cell abundance are substantially reduced in HYα and INT1-2KO mice.