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

Area of Science:

• Molecular Biology
• Immunology
• Genetics

Background:

• The Shieldin (SHLD) complex and CTC1-STN1-TEN1 (CST) complex are involved in DNA repair pathways.
• During immunoglobulin heavy-chain (Igh) class switch recombination (CSR), the 53BP1-RIF1-SHLD pathway limits DNA double-strand break (DSB) resection.
• The specific roles of the CST complex and its interaction with SHLD during CSR remain unclear.

Purpose of the Study:

• To elucidate the function of the CST complex and its interplay with the SHLD complex during immunoglobulin class switch recombination (CSR).
• To determine the necessity of CTC1 and SHLD1 in preventing DNA end resection and genetic instability during CSR.
• To investigate the functional significance of the SHLD1-CTC1 interaction in CSR.

Main Methods:

• Generation of AID-inducible B cell lines deficient for CTC1, SHLD1, or both.
• Analysis of CSR efficiency in these engineered B cell lines.
• Complementation experiments using a SHLD1 mutant defective in CST binding.

Main Results:

• CTC1-deficient B cells exhibit defects in IgM-to-IgA class switching, increased Igh chromosome breaks and translocations, and elevated DNA end resection and micro-homology usage.
• CTC1 and SHLD1 function epistatically in preventing excessive DNA end resection and genetic instability during CSR.
• A SHLD1 mutant unable to bind CST is fully functional in promoting CSR, indicating the SHLD1-CTC1 interaction is dispensable for these functions.

Conclusions:

• CTC1 is essential for suppressing alternative end-joining pathways during CSR, thereby preventing genetic instability.
• CTC1 and SHLD1 act together to limit DNA end resection and maintain genomic integrity during CSR.
• The interaction between SHLD1 and CST is not required for their respective roles in promoting CSR.