APE1 is dispensable for S-region cleavage but required for its repair in class switch recombination

Abstract

Activation-induced cytidine deaminase (AID) is essential for antibody diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ∼20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1's endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 deficiency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between Sμ and Sα. Knockdown of end-processing factors such as meiotic recombination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to facilitate the joining of broken ends during CSR.

Keywords: DNA cleavage; DNA synapse formation; class switch recombination; end processing; somatic hypermutation.

Fig. 1.

APE1's endonuclease activity is essential for CSR. (A) Schematic representation of the various APE1 mutants used in the CSR complementation experiments. (B) Representative IgA population of Ape1-null CH12F3-2A cells after transient introduction of various APE1 constructs. Data are represented as mean ± SD. (C) IgA switching efficiency of Ape1-null CH12F3-2A cells stably expressing the empty vector, WT, or Y170F mutant of APE1. APE1 protein expression was determined by Western blot (Upper), and CSR efficiency was measured by FACS as the percentage of cell surface IgA expression at indicated time after CIT stimulation (Lower). Data are represented as mean ± SD. (D) Representative flow cytometry (FACS) profile of the IgA switching population under CIT (−) and (+) conditions.

Fig. 2.

APE1 is dispensable for AID-induced 5′ Sμ mutation. Ape1-null cells stably expressing the vector, WT, or Y170F mutant of APE1 were stimulated with CIT for 72 h, followed by genomic DNA extraction for mutation analysis. (A) The diagram depicts the position of the 5′ Sμ region selected for the mutation analysis. (B) Result of the mutation analysis for the 5′ Sμ region. P values (Fisher’s exact test) for significant difference are shown in the graph. The detailed results are shown in Tables S1 and S2. (C and D) ChIP and quantitative PCR analysis for APE1 (C) and Flag (D) in cells stimulated (or not) with CIT for 24 h. Data are represented as mean ± SD.

Fig. 3.

APE1 is dispensable for AID-induced IgH/c-myc translocation. Ape1-null cells stably expressing the vector, WT, or Y170F mutant of APE1 were stimulated with CIT for 72 h, followed by genomic DNA extraction for translocation analysis. (A) A schematic representation of the PCR assay used for IgH/c-myc translocations. Primers used to detect derivative chromosome 15 (derChr15) translocations are shown as horizontal arrows. Vertical arrows represent AID-induced DNA break sites. The gray bar shows the position of the c-myc probe used for Southern detection. (B) Result of the IgH/c-myc translocation derChr15 analysis (96 lanes total from two independent experiments). P values (Fisher’s exact test) for significant difference are shown in the graph. (C) Representative Southern blots with c-myc probe for IgH/c-myc translocation are shown (10⁵ cells per lane).

Fig. 4.

AID induces a similar level of DNA break at the Sμ region in the absence of functional APE1. (A) Cells were stimulated (or not) with CIT for 24 h and then harvested for biotin-labeling DNA break assay as described in Materials and Methods. Pulled-down DNA labeled with biotin–dUTP at the cleaved ends was subjected to region-specific PCR. (B) ChIP and quantitative PCR analysis for γH2AX in cells stimulated (or not) with CIT for 24 h. Data are represented as mean ± SD. (C) LM-PCR analysis of DNA isolated from cells stimulated with CIT for 24 h. DNA was left untreated (−T4) or treated (+T4) with T4 DNA polymerase and assessed with Sμ-specific primer or by amplification of gapdh (internal control for template loading). Wedges indicate a threefold increase in DNA.

Fig. 5.

APE1 is required for efficient Sμ–Sα synapse formation during CSR. (A) Scheme of long-range interactions between Sμ–Sα elements in the IgH locus before and after AID activation. (B) Representative gel picture of the 3C assay detecting Sμ–Sα interaction in the three cell lines stimulated (or not) with CIT for 24 h. GAPDH was amplified as loading control. (C) ChIP and quantitative PCR analysis for Ku80 in cells stimulated (or not) with CIT for 24 h. Data are represented as mean ± SD.

Fig. 6.

The involvement of end-processing enzymes in the residual switching in Ape1-null CH12F3-2A cells. (A, B, and D) Protein expression (Upper) and IgA switching efficiency (Lower) of Ape1-null CH12F3-2A cells transfected with the indicated siRNA oligos and stimulated with CIT for 24 or 48 h. (C) Relative IgA switching efficiency (bar graph) and cell viability (dot plot) of CH12F3-2A–Bcl2 cells treated with various concentrations of the proteasome inhibitor Bortezomib. In all datasets, data are represented as mean ± SD.