Inactivation of GSK3β by Ser389 phosphorylation prevents thymocyte necroptosis and impacts Tcr repertoire diversity

Abstract

The assembly of Tcrb and Tcra genes require double negative (DN) thymocytes to undergo multiple rounds of programmed DNA double-strand breaks (DSBs), followed by their efficient repair. However, mechanisms governing cell cycle checkpoints and specific survival pathways during the repair process remain unclear. Here, we report high-resolution scRNA-seq analyses of individually sorted mouse DN3 and DN4 thymocytes, which reveals a G2M cell cycle checkpoint, in addition to the known G1 checkpoint, during Tcrb and Tcra recombination. We also show that inactivation of GSK3β by phosphorylation on Ser³⁸⁹ is essential for DN3/DN4 thymocytes to survive while being stalled at the G1 and G2/M checkpoints. GSK3β promotes death by necroptosis, but not by apoptosis, of DN3/DN4 thymocytes during V(D)J recombination. Failure to inactivate GSK3β in DN3 thymocytes alters the Tcrb gene repertoire primarily through Trbv segment utilization. In addition, preferential recombination of proximal V segments in Tcra depends on GSK3β inactivation. Our study identifies a unique thymocyte survival pathway, enabling them to undergo cell cycle checkpoints for DNA repair during V(D)J recombination of Tcrb and Tcra genes. Thymocyte survival during cell cycle checkpoints for V(D)J recombination DNA repair determines TCRα/β repertoire.