Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Michael X Le,Jaime F Angulo,Amit Chopra,Clare C So,Dania Haddad,Alberto Martin,Conglei Li,Rui Hu,Jason Moffat,Alexanda K Ling

doi:10.1038/srep37215

Abstract

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

Highlights

To identify novel factors involved in class switch recombination (CSR), a previously developed shRNA library[31] was introduced in bulk into the mouse B cell line, CH12F3-2, which is capable of undergoing robust CSR from IgM to IgA in vitro upon stimulation with a cocktail composed of anti-CD40, IL-4 and TGFβ(hereafter CIT)[32]
We examined our large panel of Kin shRNA – revealing that some hairpins were only capable of mild to no knockdown of the protein – with the goal of examining the relationship between Kin expression and the efficiency of CSR (Supplementary Figure S1a, Fig. 1e)
We have taken strides towards understanding the function of Kin by revealing a critical role for the protein in CSR which may reflect its involvement in DNA repair activity upstream of DSB repair pathway choice

Summary

Introduction

It remains an open question as to whether B cells require Kin function to repair the programmed DSBs generated during CSR. Surface IgA expression was measured in AID−/− CH12 cells transduced and selected for shGFP and shKin 24, transfected with the Cas[9] expression vector (px330) equipped with upstream μand downstream αguide RNAs for 48 hours. (e) Junctions from CRISPR/Cas[9] mediated CSR events in shGFP and shKin 24 AID−/− CH12 cells were sequenced and analyzed for deletion (left) and insertion length (right). Great strides have been made to advance our understanding of how programmed DSBs generated during CSR are repaired, significant knowledge gaps still remain – especially with respect to DNA damage responses that may be independent of well-studied orchestrators such as ATM or DNAPK. Our results demonstrate that Kin is required for repair of DSBs generated incidentally, as in the case of ionizing radiation, or in a programmed fashion, such as during CSR

Methods

Results

Conclusion