A DNA break- and phosphorylation-dependent positive feedback loop promotes immunoglobulin class-switch recombination

Abstract

The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.

Figure 1

Catalytically inactive AID bound to S regions is not efficiently phosphorylated. ChIP and quantitative PCR analysis of Aicda^−/− B cells retrovirally infected to express empty vector (EV) or vector encoding AID(WT), AID(S38A) or AID(DM) and stimulated for 3 d with LPS plus IL-4, assessing the abundance of histone H3, AID, p-Ser38–AID or RPA (horizontal axis) at S_μ or S_γ1 (key), presented as relative units (RU) calculated as follows: normalization of the cycling threshold (Ct) of DNA obtained by ChIP with a specific antibody to that of input DNA, followed by calculation of the inverse of that normalized Ct and subtraction of the ‘IgG preclear’ value (inverse of the normalized Ct for ChIP with IgG) from the value obtained for the ChIP with a specific antibody (inverse of the normalized Ct). *P < 0.01 and **P < 0.008, results for p-Ser38–AID or RPA in cells expressing AID(WT) versus those in cells expressing AID(DM) (paired, two-tailed Student’s t-test). Data represent three independent experiments (mean and s.d.).

Figure 2

Phosphorylation of AID at Ser38 is induced by DNA breaks. (a) ChIP and quantitative PCR analysis (as in Fig. 1) of Aicda^−/− B cells infected and stimulated as in Figure 1 and treated with ionizing radiation (10 Gy). (b) Immunoassay of Aicda^−/− B cells retrovirally infected to express empty vector or various forms of AID (above lanes) and left untreated (−IR) or treated (+IR) with ionizing radiation (10 Gy), assessed by immunoprecipitation (IP) with antibody to p-Ser38–AID covalently conjugated to agarose beads followed by immunoblot analysis of immunoprecipitates (top two) and lysates (bottom three) with anti-AID, anti-RPA or anti-GAPDH (loading control throughout). (c) ChIP and quantitative PCR analysis (as in Fig. 1) of naive wild-type BALB/c (WT), Ung^−/−Msh2^−/−, Aicda^−/− or Aicda_S38A/S38A splenic B cells stimulated for 48 h ex vivo with LPS plus IL-4. *P < 0.03, wild-type versus Ung^−/−Msh2^−/− (paired, two-tailed Student’s t-test). (d) Frequency of cells expressing surface IgG1 among naive wild-type or Apex1^+/−Apex2^−/− splenic B cells stimulated for 96 h ex vivo with LPS plus IL-4, analyzed by flow cytometry. *P < 0.04, wild-type versus Apex1^+/−Apex2^−/− (paired, two-tailed Student’s t-test). (e) ChIP analysis of wild-type and Apex1^+/−Apex2^−/− B cells stimulated as in c, presented as the ratio of the abundance of p-Ser38–AID at S_γ1 to that of AID at S_γ1. *P < 0.001 (paired, two-tailed Student’s t-test). Data represent three independent experiments (a,c–e; mean and s.d.) or are representative of four independent experiments (b).

Figure 3

Aicda^S38A/S38A B cells have fewer DSBs. (a) LM-PCR analysis of DNA from naive wild-type BALB/c, Aicda^−/− or Aicda^S38A/S38A splenic B cells stimulated ex vivo with LPS plus IL-4 and left untreated (−T4) or treated (+T4) with T4 DNA polymerase, assessed with primers specific for S_μ or by amplification of Gapdh (internal control for template loading). Wedges indicate a threefold increase in DNA. (b,c) LM-PCR analysis of DSBs in S_μ of wild-type BALB/c, Aicda^−/− or Aicda^S38A/S38A B cells stimulated with LPS alone (for analysis of CSR to IgG3; autoradiographs, Supplementary Fig. 6), LPS plus IL-4 (for analysis of CSR to IgG1), or LPS plus interferon-γ (for analysis of CSR to IgG2a), in the absence (b) or presence (c) of T4 DNA polymerase, quantified by compilation of densitometry results for autoradiographs and presented relative to the results of wild-type cells. *P < 0.001 and **P < 0.0001 (paired, two-tailed Student’s t-test). Data are representative of three experiments (a) or represent three independent experiments (b,c; mean and s.d.).

Figure 4

Aicda^S38A/S38A B cells have less colocalization of Igh with γ-H2AX foci. (a) Wide-field images of immuno-FISH of naive wild-type BALB/c, Aicda^−/− or Aicda^S38A/S38A splenic B cells stimulated ex vivo with anti-CD40 plus IL-4, assessed with a bacterial artificial chromosome probe for Igh (red) and antibody to γ-H2AX (green) and by staining of DNA with the DNA-intercalating dye DAPI (blue); yellow arrows indicate colocalization of Igh and γ-H2AX. Scale bars, 10 μm. (b) Frequency of wild-type BALB/c, Aicda^−/− or Aicda^S38A/S38A B cells (n = 100 per genotype per experiment) with colocalization of at least one Igh signal and γ-H2AX focus (as in a; actual cell numbers, Supplementary Fig. 7). *P < 0.04 (paired, two-tailed Student’s t-test). Data are representative of three independent experiments (mean and s.d. in b)

Figure 5

AID interacts with APE1. (a) Immunoassay of Aicda^−/− B cells infected to express empty vector or HA-Flag-tagged (HF-) AID(WT), AID(S38A) or AID(DM), stimulated with LPS plus IL-4 and left untreated or treated with ionizing radiation (10 Gy), assessed by immunoprecipitation of proteins from NP-40 lysates with anti-HA covalently conjugated to agarose beads followed by immunoblot analysis of immunoprecipitates (top two) and lysates (bottom three) with anti-HA, anti-APE1 or anti-GAPDH. (b) Immunoassay of Aicda^−/− B cells infected to express empty vector or untagged AID(WT), AID(S38A) or AID(DM) and stimulated and treated as in a, assessed by immunoprecipitation of proteins from NP-40 lysates with anti-APE1 followed by immunoblot analysis of immunoprecipitates and lysates with anti-AID, anti-APE1, anti-RPA or anti-GAPDH. The immunoglobulin light chain migrates very close to AID, and the two polypeptides are often difficult to resolve by immunoblot analysis. (c) Immunoassay of Aicda^−/− B cells infected, stimulated and treated as in b, assessed by immunoprecipitation of proteins from NP-40 lysates with antibody to p-Ser38-AID covalently conjugated to agarose beads followed by immunoblot analysis of immunoprecipitates and lysates with anti-AID, anti-APE1 or anti-GAPDH. (d) Immunoassay of naive wild-type, Aicda^−/−, Aicda^S38A/S38A or Ung^−/−Msh2^−/− splenic B cells stimulated for 72 h ex vivo with LPS plus IL-4 and left untreated or treated with ionizing radiation (10 Gy), assessed by immunoprecipitation of proteins from NP-40 lysates with anti-AID followed by immunoblot analysis of immunoprecipitates with anti-APE1 and of lysates with anti-APE1, anti-AID or anti-GAPDH. (e) Immunoassay of Aicda^−/− splenic B cells infected as in b and treated with 10 Gy ionizing radiation, followed by treatment of NP-40 lysates for 30 min at 25 °C with DNase I (+) or no DNase I (−) and immunoprecipitation with anti-APE1 and subsequent immunoblot analysis of immunoprecipitates and lysates with anti-AID, anti-APE1 or anti-GAPDH. Data are representative of three (a–c) or two (d,e) independent experiments.

Figure 6

ATM is required for the phosphorylation of AID bound to S regions and the interaction of AID with APE1. (a) ChIP analysis of naive wild-type BALB/c or Atm^−/− splenic B cells stimulated for 48 h ex vivo with LPS plus IL-4, presented as the ratio of the abundance of p-Ser38-AID at S_γ1 to that of AID at S_γ1. *P < 0.007 (paired, two-tailed Student’s t-test). (b) Immunoassay of naive wild-type BALB/c, Aicda^−/−, Aicda^S38A/S38A or Atm^−/− splenic B cells stimulated for 72 h ex vivo with LPS plus IL-4, assessed by immunoprecipitation of proteins from NP-40 lysates with anti-AID and immunoblot analysis of immunoprecipitates and lysates with anti-APE1, anti-AID or anti-GAPDH. Data represent three independent experiments (a; mean and s.d.) or are representative of three independent experiments (b).