Rad52 mediates class-switch DNA recombination to IgD

Abstract

In B cells, IgD is expressed together with IgM through alternative splicing of primary V_HDJ_H-Cμ-s-m-Cδ-s-m RNAs, and also through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sμ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad52 effects the 'short-range' microhomology-mediated synapsis of intra-Sμ region DSBs. Here we find that induction of IgD CSR downregulates Zfp318, and promotes Rad52 phosphorylation and recruitment to Sμ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination with extensive microhomologies, V_HDJ_H-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52^-/- B cells aborts IgD CSR in vitro and in vivo and dampens the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogates IgD CSR. Finally, Rad52 phosphorylation is associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in patients with systemic lupus erythematosus and in lupus-prone mice. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation.

Fig. 1. Expression of cell surface and secreted IgD and IgM, as well as Iμ-Cδ transcripts by alternative splicing, alternative transcription termination and CSR.

a Alternative splicing and alternative transcription termination underpin the expression of germline Iμ-Cμ and Iμ-Cδ transcripts, as well as membrane and secreted IgM and IgD in B cells. Expression of IgD stems from either Zfp318-dependent alternative mRNA splicing or Sμ–σδ CSR. In the presence of Zfp318, which represses the transcription termination sites (TTS) of the Cμ gene, mature B cells constitutively transcribe long primary V_HDJ_H-Cμ-Cδs–m transcripts initiated by the V_H promoter. These long primary transcripts undergo alternative splicing which removes intronic regions, leading to dual expression of mature V_HDJ_H-Cμs and V_HDJ_H-Cδm transcripts encoding IgM and IgD. In the absence of Zfp318, transcription stops at Cμ TTS, resulting in a shorter primary transcript, which does not contain Cδ exons, and leads to expression of a mature V_HDJ_H-Cμ−s–m transcript only. Mature B cells also transcribe Iμ, Cμ, and Cδ regions under control of the Iμ promoter. When Zfp318 is expressed, unswitched mature B cells constitutively transcribe long primary Iμ-Cμ−s−m-Cδ–s−m transcripts, which undergo alternative splicing to removes intronic regions, leading to dual expression of germline Iμ-Cμ and Iμ-Cδ transcripts. In the absence of Zfp318, Iμ promoter-initiated transcription stops at Cμ TTS, and only germline Iμ-Cμ transcripts are expressed. b Expression of Iμ-Cδ transcripts, and membrane and secreted IgD by CSR. Schematic representation of CSR from IgM to IgD. The Sμ region recombines with the σδ region and loops out the intervening DNA, which forms a switch circle. The recombined DNA is transcribed leading to expression of V_HDJ_H-Cδ−s–m and Iμ-Cδ transcripts, initiated by the V_H and Iμ promoters, respectively—in this case, Iμ-Cδ transcripts are generated as post-recombination transcripts. Graphics depict portion of the IgH locus and the resulting primary and mature transcripts. Inset depicts the detection of Sμ–σδ junctional DNA (CSR to IgD) by nested PCR amplification followed by Southern-blotting using specific Sμ and σδ probes (Southern-blotting of amplified recombined Sμ–σδ DNA from human naïve and germinal center B cells). The amplified Sμ–σδ DNA is sequenced for further analysis of the junctional sequence as well as identification and census of mutations. iEμ, IgH intronic enhancer; Iμ, intervening μ exon; μm, exon encoding the transmembrane region of IgM; δm, exon encoding the secretory piece of IgM; σδ, noncanonical switch-like region 5′ to Cδ; δs, exon encoding the secretory region of IgD; Cδm, exon encoding the transmembrane region of IgD. Dotted gray lines show splicing of primary transcripts to yield secreted and transmembrane forms of IgM and IgD.

Fig. 2. Identification of stimuli inducing CSR to IgD and Sμ–σδ junctions in mouse and human B cells.

a Unstimulated mouse (wildtype) C57BL/6 B cells and naïve Aicda^−/− B cells (nil), or C57BL/6 and naïve Aicda^−/− B cells stimulated for 96 h with LPS, LPS plus IL-4, LPS plus TGF-β and RA, CD154, CD154 plus IL-4, CD154 plus TGF-β and RA, CpG alone, CpG plus IL-4, CpG plus TGF-β and RA, R848 alone, R848 plus IL-4, R848 plus TGF-β and RA, or CpG plus IL-4, or R848 plus IL-4 in the presence of anti-BCR Ab were analyzed for recombined Sμ–σδ, as well as recombined Sμ–Sγ1, Sμ–Sγ3, Sμ–Sα, and Sμ–Sα DNA by nested PCR using forward Iμ and reverse Cδ primers, or forward Iμ and reverse Sγ1, Sγ3, Sα or Sε primers, respectively, followed by Southern-blotting using a specific Sμ, σδ, Sγ1, Sγ3, Sα, or Sε probe, as indicated. b Recombined Sμ–σδ DNA in human tonsil IgD⁺ B cells, blood naive IgM⁺IgD⁺ B cells, or blood naive IgM⁺IgD⁺ B cells stimulated with CD145 or CpG plus IL-2 and IL-21, IL-4 and IL-21, or IL-2, IL-4 and IL-21, or IL-15 and Il-21, were analyzed 120 h post-stimulation by nested PCR using forward Iμ and reverse Cδ primers, followed by Southern-blotting using specific human Sμ or σδ probe. Data are one representative of three independent experiments yielding comparable results. c Quantification of germline/post-recombination Iμ-Cδ transcripts, post-recombination Iμ-Cγ1, Iμ-Cα, and Iμ-Cε transcripts in wildtype C57BL/6 B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, analyzed 72 h post-stimulation by qRT-PCR and normalized to β-Actin expression. Each dot represents data obtained with B cells from an individual mouse (n = 3 per group). Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 3. Mouse and human Sμ–σδ DNA recombination junctions contain microhomologies and somatic mutations.

a Amplified DNAs from junctional intra-σδ deletions as well as Sμ–σδ, Sμ–Sγ1 and Sμ–Sα1 recombinations from human tonsil B cells or human peripheral blood naïve IgM⁺IgD⁺ B cells stimulated with CpG plus IL-2 and IL-21 and cultured for 120 h, OVA-immunized C57BL/6 mouse spleen B cells or C57BL/6 mouse naïve IgM⁺IgD⁺ B cells stimulated with LPS plus IL-4 and cultured for 96 h were amplified and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in intra-σδ deletions, Sμ–σδ, Sμ–Sγ1, and Sμ–Sα1 junctional DNAs are shown by violin plots. Each dot represents a unique junctional sequence (n = 45 per group). b Human and mouse Sμ and σδ regions consist of repetitive motifs, which are better-suited substrates for Rad52-mediated MMEJ than those in Sμ and Sγ1 or Sμ and Sα. As such, they can facilitate the formation of microhomologies. Repetitive sequence elements in mouse and human Sμ, σδ, Sγ1 and Sα that can potentially form microhomologies were identified by Pustell Matrix dot plot using MacVector software and are depicted by small dots. Intensity of dots depicts frequency and degree of complementarity of respective sequences. c Somatic point-mutations in Sμ and σδ regions abetting recombined Sμ−σδ DNA junctions in IgD class-switched human and mouse B cells in vivo and in vitro. Mutations were identified in a 48–506 nt stretch of Sμ or σδ regions in unique Sμ–σδ DNA recombination sequences. Each dot represents an individual sequence. Sequence data were pooled from three individuals in each group. Box and whiskers plots show median, quartiles, maximum and minimum of mutation frequencies in Sμ and σδ regions. In pie charts, the size of slices denotes the proportion of transcripts with the same number of mutations and the gray hue denotes the number of point mutations per transcript. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). **p < 0.01, ***p < 0.001, ns: not significant (unpaired two-tailed t-test). Source data are provided as a Source Data file.

Fig. 4. Rad52 mediates Sμ–σδ DNA recombination leading to IgD secretion.

a Recombined Sμ–σδ, Sμ−Sγ1, Sμ−Sα, and Sμ−Sε DNAs in mouse Rad52^+/+, Rad52^−/− and Aicda^−/− naïve IgM⁺IgD⁺ B cells stimulated with nil, LPS alone, LPS plus IL-4, LPS plus TGF-β and RA, CD154 alone, CD154 plus IL-4, or CD154 plus TGF-β and RA, as well as Sμ–Sγ3 in Rad52^+/+, Rad52^−/− and Aicda^−/− B cells stimulated with LPS only, were analyzed 96 h post-stimulation by specific nested PCR using forward Iμ and reverse Cδ, Sγ1, Sγ3, Sα, or Sε primers, respectively, followed by Southern-blotting using specific Sμ, σδ, Sγ1, Sγ3, Sα, or Sε probe, as indicated. Data are one representative of three independent experiments yielding comparable results. b IgD titers in culture (96 h) fluid of Rad52^+/+, Rad52^−/− or Aicda^−/− B cells stimulated with LPS plus IL-4, as measured by dot-blotting (two-fold serial diluted culture fluid) using a rat anti-mouse IgD mAb. Data are one representative of five independent experiments yielding comparable results. Source data are provided as a Source Data file.

Fig. 5. Rad52 deletion ablates in vivo Sμ–σδ DNA recombination and reduces IgD production.

Rad52^+/+ and Rad52^−/− mice were immunized with OVA in alum i.p. a Recombined Sμ–σδ, Sμ−Sγ1, and Sμ−Sα DNAs in spleen, mesenteric lymph nodes (MLNs), and Peyer’s patches (PPs) B cells, as analyzed by nested PCR using forward Iμ and reverse Cδ, Sγ1, or Sα primers, respectively, followed by Southern-blotting using specific Sμ, σδ, Sγ1, or Sα probe, as indicated. Data are one representative of three independent experiments yielding comparable results. b Sμ–σδ, Sμ–Sγ1, and Sμ–Sα junctional DNAs were amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sμ–σδ, Sμ−Sγ1, and Sμ−Sα junctional DNAs are shown by violin plots. Each symbol represents a unique sequence (n = 45 per group). c–f Titers of total IgD in serum, BALF, and feces, as analyzed by dot-blotting using rat anti-mouse IgD mAb—titers of total IgM, IgD, IgG1, and IgA as well as OVA-binding IgM, IgD, IgG1, and IgA as analyzed by specific ELISAs. Each dot represents datum from one individual mouse (n = 5–8 per group, as indicated). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (unpaired two-tailed t-test). No adjustments were made for multiple comparisons. g Bacteria-bound IgD and IgA in feces as analyzed by flow cytometry. h IgM, IgD, and IgA positive cells in MLNs and lamina propria as visualized by fluorescence microscopy. Scale bar = 100 μm. Data in g, h are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 6. Rad52 is phosphorylated and recruited to Sμ and σδ in B cells induced to undergo IgD CSR.

a C57BL/6 mouse naïve IgM⁺IgD⁺ B cells were stimulated with LPS plus IL-4 and cultured for 0, 24, 48, 72 and 96 h. Rad52, Ku70, Ku86, and Aicda transcripts were analyzed by real-time qRT-PCR, normalized to β-Actin expression, and depicted as relative to the expression in unstimulated B cells (set as 1.0). Data are mean ± SEM of three independent experiments. b Expression of Rad52, phosphorylated Rad52 (p-Rad52), AID, Ku70, Ku86, and β-Actin proteins in mouse B cells stimulated with LPS plus IL-4 (as in a), as analyzed by specific immunoblotting. Data are one representative of three independent experiments yielding comparable results. c Human peripheral blood naive IgM⁺IgD⁺ B cells were stimulated with CD154 plus IL-4 and IL-21 and cultured for 0, 24, 48, 72 and 96 h. RAD52, KU70, KU86, and AICDA transcripts were analyzed by real-time qRT-PCR, normalized to β-ACTIN expression, and depicted as relative to the expression in unstimulated B cells (set as 1.0). Data are mean ± SEM of three independent experiments. d Recruitment of Rad52 to σδ region DNA, as analyzed by ChIP-qPCR assays in mouse Rad52^+/+ and Rad52^−/− B cells stimulated with LPS plus IL-4 and cultured for 72 h. Data are expressed as percent of pre-IP input for each sample (mean ± SEM of three independent experiments). ***p < 0.001 (unpaired two-tailed t-test). e, f C57BL/6 mouse naïve IgM⁺IgD⁺ B cells were stimulated with nil, LPS alone, LPS plus IL-4, or LPS plus TGF-β and RA and cultured for 72 h. Recruitment of Rad52 (e) and Ku70/Ku86 (f) to Sμ, σδ, Sγ1, Sγ3, and Sα region DNA, as analyzed by ChIP-qPCR assays. Data are mean ± SEM of three or four independent experiments. Source data are provided as a Source Data file.

Fig. 7. Stimuli inducing Sμ–σδ DNA recombination downregulate ZFP318/Zfp318 in human and mouse B cells.

a C57BL/6 mouse naïve IgM⁺IgD⁺ B cells were stimulated with nil, LPS plus IL-4 or LPS plus TGF-β and RA. Surface expression of IgM and IgD were analyzed 96 h post-stimulation by flow cytometry. Expression of V_HDJ_H−Cδm, V_HDJ_H−Cδs, V_HDJ_H−Cμm, and V_HDJ_H−Cμs transcripts were analyzed 72 h post-stimulation by semi-quantitative RT-PCR using serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. b IgD in supernatant from cultures (96 h) of C57BL/6 naïve IgM⁺IgD⁺ B cell stimulated with nil, LPS plus IL-4, LPS plus TGF-β and RA, or CD154 plus IL-4, as analyzed by dot-blotting using rat anti-mouse IgD mAb. Data are representative of 5 independent experiments. c Expression of Zfp318 transcripts in mouse naïve B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by qRT-PCR and normalized to β-Actin expression and depicted relative to the average expression in unstimulated B cells (set as 1). Data are mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (unpaired two-tailed t-test). d Expression of Zfp318 transcripts in unstimulated mouse naïve B cells (Nil) and mouse naïve B cells stimulated with LPS plus IL-4 for 72 h, as analyzed by mRNA-Seq. Data are mean ± SEM of four independent experiments. ***p < 0.001 (unpaired two-tailed t-test). e Zfp318 protein level in mouse naïve B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 96 h post-stimulation by intracellular staining with rabbit anti-Zfp318 Ab in flow cytometry. Bars in the right panel represent level of MFI (mean ± SEM) from three independent experiments. *p < 0.05, ***p < 0.001 (unpaired two-tailed t-test). f Human blood naïve IgM⁺IgD⁺ B cells were stimulated with nil, CpG plus IL-2 and IL-21 or CpG plus IL-4 and IL-21; V_HDJ_H−Cδm, V_HDJ_H−Cδs, V_HDJ_H−Cμm, and V_HDJ_H−Cμs transcript levels were measured 72 h post-stimulation by semi-quantitative RT-PCR with serial two-fold dilution of cDNA templates—data are representative of three independent experiments (left panels). Expression of ZFP318 transcripts as analyzed 72 h post-stimulation by qRT-PCR and normalized to HPRT expression (f middle panel)—data are mean ± SEM of three independent experiments. Secreted IgD in supernatants of the human B cell cultures, as analyzed 120 h post-stimulation by specific ELISA (f right panel)—data are mean ± SEM of four independent experiments. **p < 0.01, ***p < 0.001 (unpaired two-tailed t-test). g Expression of ZFP318 transcripts in human naïve CD27⁻IgM⁺IgD⁺ B cells and memory CD27⁺IgM⁻IgD⁺ B cells isolated from peripheral blood of healthy subjects, as analyzed by mRNA-Seq. Data are mean ± SEM of three independent experiments. h Expression of V_HDJ_H−Cδm, V_HDJ_H−Cδs, V_HDJ_H−Cμm, and V_HDJ_H−Cμs transcripts in human tonsil IgD⁺ B cells, as analyzed by semi-quantitative RT-PCR involving serial two-fold dilution of cDNA templates (left panel)—data are representative of three independent experiments. Expression of ZFP318 protein in human tonsil IgM⁺IgD⁺ B cells and IgM⁻IgD⁺ B cells, as analyzed by intracellular staining with anti-Zfp318 Ab in flow cytometry (middle panel)—data are representative of four independent experiments (mean ± SEM, right panel). **p < 0.01, ns: not significant (unpaired two-tailed t-test). i Surface expression of IgM and IgD in mouse naïve Rad52^+/+, Rad52^−/− and Aicda^−/− B cells stimulated with LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 96 h post-stimulation by flow cytometry. Data are representative of three independent experiments. j Expression of V_HDJ_H−Cδm and V_HDJ_H−Cδs transcripts in mouse naïve Rad52^+/+ and Rad52^−/− B cells stimulated with nil, LPS plus IL-4 or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by semi-quantitative RT-PCR using serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. k Rad52 or AID deficiency does not alter Zfp318 expression. Expression of Zfp318 transcripts in mouse naïve Rad52^+/+, Rad52^−/− and Aicda^−/− B cells stimulated with nil, LPS plus IL-4, or LPS plus TGF-β and RA, as analyzed 72 h post-stimulation by qRT-PCR and normalized to β-Actin expression, as depicted relative to expression in unstimulated B cells (set as 1). Data are mean ± SEM of three independent experiments. No adjustments were made for multiple comparisons. Source data are provided as a Source Data file.

Fig. 8. RAD52 is required for Sμ–σδ DNA recombination in human B cells.

a Human blood naïve IgM⁺IgD⁺ B cells were transfected with specific RAD52 siRNA or scrambled (Scra) siRNA and stimulated by CpG plus IL-2 and IL-21. Recombined Sμ–σδ and Sμ–Sγ1 DNA in the transfected B cells 120 h after RAD52 siRNA transfection, as well as Sμ–σδ DNA in tonsil IgM^–IgD⁺ and blood naive IgM⁺IgD⁺ B cells were analyzed by nested PCR using forward Iμ and reverse Cδ or Sγ1 primers followed by Southern-blotting using indicated specific probes. Data are from three independent experiments. b Expression of RAD52 and AICDA transcripts was analyzed 48 h after RAD52 siRNA or Scra siRNA transfection by qRT-PCR and normalized to HPRT expression. Data are mean ± SEM of three independent experiments. **p < 0.01, ns: not significant (unpaired two-tailed t-test). c Expression of RAD52 and AID proteins were analyzed 72 h after RAD52 siRNA or Scra siRNA transfection by specific Western blotting. Data are representative of three independent experiments. d Expression of V_HDJ_H−Cδm and V_HDJ_H−Cδs transcripts as analyzed by semi-quantitative RT-PCR with of serial two-fold dilution of cDNA templates. Data are representative of three independent experiments. e RAD52 is recruited to σδ region DNA in human B cells undergoing CSR to IgD. Recruitment of RAD52 to Sμ and σδ region DNA in human blood naive IgM⁺IgD⁺ B cells stimulated for 120 h with CpG plus IL-2 and IL-21, as analyzed by specific ChIP-qPCR. Data are mean ± SEM of three or four independent experiments. *p < 0.05, **p < 0.01 (unpaired two-tailed t-test). Source data are provided as a Source Data file.

Fig. 9. B cells undergoing CSR to IgD differentiate to IgD-producing plasmablasts/plasma cells.

a Human blood naive IgM⁺IgD⁺ B cells were stimulated with CpG plus IL-2 and IL-21, which induce IgD CSR, or CpG plus IL-4 and IL-21, which do not induce IgD CSR. Proportions of CD138⁺IgM⁻IgD⁺ plasmablasts/plasma cells among intracellular sIgM⁻IgD⁺ B cells and BLIMP-1 expression in intracellular sIgM^–IgD⁺ cells, as analyzed 120 h post-stimulation by flow cytometry. Alexa Fluor 647-fluorescence minus one (FMO) controls are shown as reference. b Mouse Rad52^+/+ B cells and Rad52^−/− B cells stimulated with LPS plus IL-4, which induce IgD CSR. Proportions of sCD138⁺ plasmablasts/plasma cells among intracellular IgD⁺sIgM⁻ cells and Blimp-1 expression in intracellular IgD⁺sIgM⁻ cells, as analyzed 96 h post-stimulation by flow cytometry. FITC-FMO controls are shown as reference. Data in a and b are representative of three independent experiments. c Recombined Sμ–σδ and Sμ–Sα DNA in two IgD⁺ myelomas and one IgA⁺ myeloma, as analyzed by specific nested PCR followed by Southern-blotting using indicated probes.

Fig. 10. p-Rad52 expression, CSR to IgD and antinuclear antigen IgD autoantibodies in lupus patients and mice.

a Serum total and double-strand DNA (dsDNA)-, RNA-, histone-, or RNP/Sm-binding IgD in healthy human subjects and systemic lupus erythematosus (SLE) patients, as analyzed by specific ELISAs. Each dot represents the datum from one individual human subject. Mean ± SEM of 6–10 healthy subjects or SLE patients are depicted. *p < 0.05 (unpaired two-tailed t-test). b Human and mouse antinuclear autoantibodies (ANAs), as visualized by indirect immunofluorescence microscopy on HEp-2 cells that were incubated with serum from a healthy human subject, an SLE patient, a C57BL/6 mouse, or a MRL/Fas^lp/lpr mouse, as revealed by FITC-labeled rat mAb to human or mouse IgD. Scale bar = 50 μm. c Total IgD in serum, feces and BALF as analyzed by dot-blotting, and concentrations of IgD autoantibodies to dsDNA or histone in serum of C57BL/6 and MRL/Fas^lpr/lpr mice, as analyzed by specific ELISAs. Each dot represents datum from one individual mouse. Data are mean ± SEM of 3–9 mice, as indicated. **p < 0.01, ***p < 0.001 (unpaired two-tailed t-test). d IgD concentrations in serum, feces and BALF from C57BL/6 and MRL/Fas^lpr/lpr mice, as analyzed by dot-blots. Shown are dot-blots from one C57BL/6 and one MRL/Fas^lpr/lpr mouse, representative of 3–9 C57BL/6 and MRL/Fas^lpr/lpr mice. e Bacteria-bound IgD and IgA in feces from C57BL/6 and MRL/Fas^lpr/lpr mice, as analyzed by flow cytometry. f Expression of V_HDJ_H−Cδm, V_HDJ_H−Cδs, V_HDJ_H−Cμm, and V_HDJ_H−Cμs transcripts in bone marrow (BM), spleen and mesenteric lymph nodes (MLNs), as analyzed semi-quantitative RT-PCR by serial two-fold dilutions of cDNA templates. Shown are RT-PCR data from one C57BL/6 mouse and one MRL/Fas^lpr/lpr mouse, representative of three C57BL/6 and three MRL/Fas^lpr/lpr mice. g IgD⁺ B cells in lamina propria, MLNs and Peyer’s patches (PPs) of C57BL/6 and MRL/Fas^lpr/lpr mice, as visualized by fluorescent microscopy. Scale bar =  100 μm. h Recombined junctional Sμ–σδ, Sμ–Sγ1, and Sμ–Sα DNAs in bone marrow, spleen, MLNs, and PPs B cells from C57BL/6 and MRL/Fas^lpr/lpr mice as analyzed by specific nested PCR using forward Iμ and reverse Cδ, Sγ1 or Sα primers, followed by Southern-blotting using indicated probes. Data are representative of three independent experiments. i Sμ–σδ, Sμ–Sγ1 and Sμ–Sα junctional DNAs in non-immunized MRL/Fas^lpr/lpr mice, as amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sμ–σδ, Sμ–Sγ1, and Sμ–Sα junctional DNAs are rendered by violin plots. Each dot represents a unique sequence (n = 45 per group). j Somatic point-mutations in Sμ and σδ regions abetting recombined Sμ−σδ DNA junctions in IgD class-switched spleen B cells from three MRL/Fas^lpr/lpr mice. Mutations were identified in a 48–506 nt stretch of Sμ or σδ regions in unique Sμ–σδ DNA recombination sequences. Each dot represents an individual sequence. ns: not significant (unpaired two-tailed t-test). Box and whiskers plots show the median, quartiles, maximum, and minimum of mutation frequencies in Sμ and σδ regions. In pie charts, the size of slices denotes the proportion of sequences with the same number of mutations and the gray hue denotes the number of point-mutations per sequence. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). k Expression of phosphorylated Rad52 (p-Rad52), Rad52 and β-Actin proteins in peripheral blood B cells from healthy human subjects and SLE patients as well as B cells from C57BL/6 mice and MRL/Fas^lpr/lpr mice, as analyzed by specific Western blotting using rabbit anti-p-Rad52 Ab or anti-β-Actin mAb—p-Rd52 (Y104) Ab detected endogenous levels of Rad52 protein only when phosphorylated at tyrosine 104. l Expression of ZFP318 and AICDA transcripts in B cells from healthy human subjects and SLE patients (left panel), as well as Zfp318 and Aicda transcripts in MLNs from C57BL/6 and MRL/Fas^lpr/lpr mice (right panel), as analyzed by specific qRT-PCR. Data are mean ± SEM of three healthy human subjects, three SLE patients, three C57BL/6 mice, and three MRL/Fas^lpr/lpr mice. *p < 0.05 (unpaired two-tailed t-test). No adjustments were made for multiple comparisons. Source data are provided as a Source Data file.