Class switch recombination junctions are not affected by the absence of the immunoglobulin heavy chain E_μ enhancer

After encountering antigen, B-cells undergo class switch recombination (CSR) that substitutes the constant (C)_μ gene with C_γ, C_ε, or C_α, thereby generating IgG, IgE, and IgA antibodies with new effector functions but the same antigenic specificity.¹ The DNA-editing enzyme activation-induced deaminase (AID) is required for CSR by targeting specific DNA switch (S) regions preceding the C region, except C_δ.² S_μ is the donor region, while S_γ,ε,α are the acceptor regions. CSR is controlled in cis by the immunoglobulin heavy chain (IgH) 3’regulatory region (3’RR).³ The 3’RR is essential to poise AID on the S acceptor region. During CSR IgH, intrachromosomal interactions (schematized in Fig. 1a) are found between the 3’RR and the intronic E_μ enhancer.^1,4 Looping allows transcriptional binding activators to enhancers to facilitate CSR. However, CSR is only modestly influenced by E_μ deletion.^5–7 During CSR, two different DNA repair pathways take place: the classical nonhomologous end joining (c-NHEJ) and the alternative end joining (A-EJ) pathways. The c-NHEJ pathway (which uses components such as 53BP1, Ku70, ku80, XRCC4, and ligase 4) is preferentially implicated for blunt and microhomology structure junctions and the A-EJ pathway (which uses components such as CtIP, MRN, PARP1, DNA Pol θ) favors junctions with large homology.^8,9 Data have reported that when an element of the DNA damage response pathway is defective, the molecular signature of CSR junctions is affected, suggesting that junctions are repaired through a different DNA repair pathway.¹⁰ We recently reported a new computational tool (CSReport) for the automatic analysis of CSR junctions sequenced by high-throughput sequencing,¹¹ and used it to analyze the rare S_μ-S_δ junctions during IgD CSR,^12,13 S_μ-S_γ,α junctions in wild-type (wt) mice¹⁴ and UNG-deficient mice.¹⁵ Even if E_μ-deficient mice exhibited a modest defect in IgH CSR, it is conceivable that its participation in CSR 3D loop interactions would be important for the DNA repair process. We used the CSReport and high-throughput sequencing to analyze the molecular signature of S_μ-S_γ3, S_μ-S_γ1, and S_μ-S_α junctions in E_μ-deficient mice to perform a more in depth search for a putative difference in their DNA repair pathways compared to those in wt mice.

Fig. 1.

Class switch recombination in Eμ-deficient mice. a Schematic representation of IgH intrachromosomal interactions between E_μ and 3’RR transcriptional enhancers with the S acceptor and S donor regions during CSR (according to references 1 and 4). b IgG₁, IgG₃, and IgA CSR in E_μ-deficient and wt mice. B-splenocytes were LPS-cultured for 4 days with (CSR toward IgG₁) or without (CSR toward IgG₃) the addition of IL-4 or TGFβ (CSR toward IgA). Cells gated on B220⁺ and/or CD138⁺ cells were investigated with anti-IgM, anti-IgG₃, anti-IgG₁, and anti-IgA antibodies. One representative experiment out of a total of 3–5 experiments for each isotype is shown. Mean ± SEM of three to five experiments for each isotype is reported. Significances were investigated using the Mann–Whitney U-test. c Structure profiles of S_μ-S_γ1, S_μ-S_γ3, and S_μ-S_α junctions in E_μ-deficient and wt mice. Junctions are classified in terms of junction with insertion, blunt junction, junction with microhomology (<4 bp) or large homology (≥4 bp). The junction profile was not significantly different in wt and E_μ-deficient mice. The results are pooled from three to seven mice. d Breakpoint localization in S_μ-S_γ1, S_μ-S_γ3, and S_μ-S_α junctions in wt and E_μ-deficient mice (same junctions as in Fig. 1c). e Motif targeting in S_μ-S_γ1, S_μ-S_γ3, and S_μ-S_α junctions. The stars identify breakpoint positions in AGCT, WRCY, and RGYW AID hotspots (same junctions as shown in Fig. 1c)

The mice were housed and the procedures were conducted in agreement with European Directive 2010/63/EU on animals used for scientific purposes applied in France as the « Décret n°2012–118 du 1^er février 2013 relatif à la protection des animaux utilisés à des fins scientifiques ». Accordingly, the present project (APAFiS≠13855) was authorized by the « Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche ». E_μ-deficient and wt mice (housed in a conventional animal facility) were used. Single-cell suspensions of B-splenocytes were cultured for 4 days at 1 × 10⁶ cells/ml in RPMI 1640 supplemented with 10% fetal calf serum and 5 μg/ml LPS, with (CSR toward IgG₁) or without (CSR toward IgG₃) the addition of 20 ng/ml IL-4 or 2 ng/ml TGFβ (PeproTech, Rocky Hill, NJ) (CSR toward IgA).^14,16 Splenocytes were washed in PBS and stained with various antibodies: anti-B220-BV510 (BioLegends), anti-CD138-APC (Becton Dickinson Biosciences), anti-IgM PECy7 (ebiosciences), anti-IgG₃-FITC (Becton Dickinson Biosciences), anti-IgG₁-PE (Becton Dickinson Biosciences), and anti-IgA-FITC (Southern Biotech). The cells were analyzed on a Fortessa LSR2 (Beckman Coulter). In parallel experiments, stimulated B-cell splenocyte DNA was extracted for the investigation of S_μ-S_γ3, S_μ-S_γ1, and S_μ-S_α junctions. As previously described in detail,¹¹ the junctions were PCR amplified. Libraries of 200 bp were prepared from the 1-2 kb PCR products of S_μ-S_γ3, S_μ-S_γ1, and S_μ-S_α amplification for Ion Proton sequencing (“GénoLim platform” of the Limoges University, France). The sequenced reads were then mapped to S_μ, S_γ1, S_γ3, and S_α regions using the BLAST algorithm. As previously reported,¹¹ the computational tool developed for experiments performs junction assembly, identifies breakpoints in S_μ, S_γ1, S_γ3, and S_α, identifies junction structure (blunt, microhomology, large homology or insertion junctions) and outputs a statistical summary of the identified junctions.

Confirming previous studies,^5–7 flow cytometry experiments indicated that compared to wt mice, the deletion of the E_μ enhancer had only a minor impact on CSR toward IgG₃ (mean 5.0 vs 5.6%), IgG₁ (mean 8.5 vs 19.3%), and IgA (mean 1.7 vs 3.5%) (Fig. 1b). We then extracted stimulated B-cell DNA to investigate the molecular signatures of these S_μ-S_γ3 (IgG₃ CSR), S_μ-S_γ1 (IgG₁ CSR), and S_μ-S_α (IgA CSR) junctions. When a few dozen CSR junctions are reported after cloning and subsequent Sanger sequencing, several hundred or thousand junctions can be documented using our computational tool of CSR junctions sequenced by high-throughput sequencing. The structural profiles (blunt, microhomology, large homology or junction with insertions) of 2443 S_μ-S_γ3, 8982 S_μ-S_γ1, and 617 S_μ-S_α E_μ-deficient junctions and 7209 S_μ-S_γ3, 14,700 S_μ-S_γ1, and 2639 S_μ-S_α wt junctions are reported in Fig. 1c. No significant difference (Mann–Whitney U-test) was found between E_μ-deficient mice and wt mice. In addition to the type of junctions, the distribution of IgG₃, IgG₁, and IgA junctions in terms of distance from the forward PCR primer in S_μ and from one of the reverse primers in S_γ3, S_γ1, and S_α was similar in E_μ-deficient and wt mice (Fig. 1d). The same results were found for the localization of the breakpoints within AID hotspots (AGCT, WRCY, RGYW) and other motifs (Fig. 1e) (displayed along specifically targeted segments within S regions).

As previously reported by others,^5–7 the efficacy of CSR is, at most, modestly reduced by the deletion of the E_μ enhancer. Until recently, the transcriptional enhancers located in the IgH 3’RR are the sole cis-regulatory elements reported to affect conventional CSR.^2,3,17 Compared with wt mice, the type and location of the CSR junctions in the absence of E_μ are unaffected. This observation is highlighted with three different isotypes (γ1, γ3, and α) by the analysis of a thousand independent junctions using our computational tool of CSR junctions sequenced by high-throughput sequencing. The E_μ enhancer is dispensable to poise AID to S donor/acceptor regions and for the generation of double strand breaks.¹⁸ E_μ IgH intrachromosomal interactions with S donor/acceptor regions and the 3’RR do not seem important to recruit/stabilize DNA repair factors and, thus, for the choice of the DNA repair pathway. In fact, do these E_μ IgH intrachromosomal interactions have a real relevance? Are these interactions only mechanical due to the IgH location of E_μ and to the 3D folding that brings S regions into contact with each other? These questions remain open. Deletion of the cis-regulatory IgH 3’RR affects CSR to all isotypes (except IgD).^3,17 We currently investigate the molecular signature of the remaining CSR junctions in 3’RR-deficient mice to search for a potential role in the recruitment of specific DNA repair factors.

Author contributions

Y.D. designed the research. N.G., H.I., and M.F. performed the research. F.B. and Y.D. analyzed the data. N.G., H.I., M.F., F.B., and Y.D. wrote the paper.

Competing interests

The authors declare no competing interests.