CST Is Epistatic With Shieldin to Limit DNA Double-Strand Break End Resection and Promote Repair During Igh Class Switch Recombination

Abstract

Downstream of 53BP1-RIF1 lies the Shieldin (SHLD) protein complex, which comprises MAD2L2/REV7, SHLD3, SHLD2, and SHLD1, and the CTC1-STN1-TEN1 (CST) complex. During immunoglobulin heavy-chain (Igh) class switch recombination (CSR), 53BP1-RIF1-SHLD promotes productive end-joining by limiting resection of activation-induced cytidine deaminase (AID)-generated DNA double-strand break (DSB) ends. The precise role of the CST complex and its interplay with SHLD during CSR is however elusive. Here, we established AID-inducible B cell lines deficient for CTC1, SHLD1, or both and analyzed CSR in these cells. We show that stimulated CTC1-deficient B cells are defective for IgM-to-IgA class switching, accumulate Igh chromosome breaks and translocations, and display increased end-resection and micro-homology usage at switching sites, demonstrating that CTC1 is essential to suppress alternative end-joining during CSR. We show that CTC1 and SHLD1 are epistatic in preventing exacerbated DNA end resection and genetic instability during CSR. Moreover, using a complementation approach in Shld1 knockout splenic B cells, we show that a SHLD1 mutant defective in CST binding (SHLD1^ΔLDLP) is fully proficient for IgM-to-IgG1, IgG2b, IgG3, and IgA class switching, thus demonstrating that the SHLD1-CTC1 interaction through this motif is dispensable for CST and SHLD functions in promoting CSR.