SHLD 2 promotes class switch recombination by preventing inactivating deletions within the Igh locus
2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês
10.15252/embr.201949823
ISSN1469-3178
AutoresAlexanda K. Ling, Meagan Munro, Natasha Chaudhary, Conglei Li, Maribel Berrú, Brendan W. Wu, Daniel Durocher, Alberto Martín,
Tópico(s)Transgenic Plants and Applications
ResumoArticle17 June 2020free access Transparent process SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus Alexanda K Ling Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Meagan Munro Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Natasha Chaudhary Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Conglei Li Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Maribel Berru Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Brendan Wu orcid.org/0000-0002-2892-864X Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Daniel Durocher orcid.org/0000-0003-3863-8635 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Alberto Martin Corresponding Author [email protected] orcid.org/0000-0002-0795-0418 Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Alexanda K Ling Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Meagan Munro Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Natasha Chaudhary Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Conglei Li Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Maribel Berru Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Brendan Wu orcid.org/0000-0002-2892-864X Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Daniel Durocher orcid.org/0000-0003-3863-8635 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Alberto Martin Corresponding Author [email protected] orcid.org/0000-0002-0795-0418 Department of Immunology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Author Information Alexanda K Ling1, Meagan Munro2, Natasha Chaudhary2, Conglei Li1, Maribel Berru1, Brendan Wu1, Daniel Durocher2,3 and Alberto Martin *,1 1Department of Immunology, University of Toronto, Toronto, ON, Canada 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada 3Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada *Corresponding author. Tel: +1 416 978 4230; E-mail: [email protected] EMBO Rep (2020)21:e49823https://doi.org/10.15252/embr.201949823 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2−/− mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2−/− primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus. Synopsis The recently described Shieldin complex inhibits DNA resection. This study shows that Shieldin deficiency rescues BrcaΔ11 embryonic lethality and impairs class switch recombination in primary B-cells through loss of immunoglobulin coding sequence as a result of enhanced DNA resection. Shld2−/− primary B-cells have impaired ex vivo and in vivo CSR. A significant and heretofore undescribed Iglo population presents during CSR in Shieldin- and other DNA repair-deficient B-cells. Iglo cells are Ig-negative as a result of hyper-resection into the donor Ig constant region. Introduction Across multiple phyla, adaptive immunity requires programmed DNA damage and concomitant repair to generate antigen receptor diversity and fine-tune the immune response. These phenomena are also particularly well suited for studying the various DNA repair pathways responsible for maintaining genome integrity. In particular, the humoral response mediated by B-cells requires the non-homologous end-joining (NHEJ) repair pathway for V(D)J recombination of the antigen-binding immunoglobulin (Ig) variable region, as well as class switch recombination (CSR) of the Ig constant regions (Kenter, 2005). Activation-induced cytidine deaminase (AID) plays a central role in humoral immunity by inducing somatic hypermutation (SHM) and CSR that function to increase the antibody affinity and alter the antibody isotype, respectively (Muramatsu et al, 2000). AID accomplishes these processes by deaminating dC to produce dU within the immunoglobulin DNA encoding the variable region and the non-coding switch regions (Martin et al, 2002; Petersen-Mahrt et al, 2002; Bransteitter et al, 2003). At this point, both processes diverge: In the context of SHM, damage produced by AID at the variable region is engaged by base excision repair and mismatch repair pathways leading to small mutations that may potentially increase the affinity of the antibody to antigen (Cascalho et al, 1998; Wiesendanger et al, 2000; Di Noia & Neuberger, 2002; Martin et al, 2003; Rada et al, 2004; Wilson et al, 2005). By contrast in CSR, damage produced by AID at the switch μ (Sμ) and downstream switch regions (Sγ, Sε, and Sα) is engaged by the same DNA repair pathways, with the distinction that these lesions are converted to double-stranded DNA breaks (DSBs), followed by synapsis and ligation of distant ends through NHEJ or the poorly defined alternative end joining (Pan et al, 2002; Kenter, 2005; Masani et al, 2016). The process of CSR allows for the expression of a novel downstream Ig constant region, with differing effector functions, with the pre-existing variable region. The joining reaction during CSR requires the NHEJ pathway. One member, 53BP1, plays a key role in CSR (Manis et al, 2004) by inhibiting DNA end resection (Bothmer et al, 2010; Bunting et al, 2010), thereby shuttling the repair of DSBs toward NHEJ instead of the homologous recombination pathway. Downstream of 53BP1 lies RIF1 that further facilitates DNA end protection and is necessary for CSR (Chapman et al, 2013; Di Virgilio et al, 2013; Escribano-Diaz et al, 2013; Zimmermann et al, 2013). The recently identified shieldin complex, that lies immediately downstream of the 53BP1-RIF1 axis, is necessary for CSR, NHEJ, and telomere protection (Dev et al, 2018; Findlay et al, 2018; Ghezraoui et al, 2018; Gupta et al, 2018; Noordermeer et al, 2018; Tomida et al, 2018). Shieldin is composed of REV7, SHLD1, SHLD2, and SHLD3. SHLD2 has three OB-fold domains that bind to single-stranded DNA (Noordermeer et al, 2018; Setiaputra & Durocher, 2019), much like RPA1 and POT1 proteins, and appears to be the factor proximally responsible for the end protection role ascribed to the 53BP1 pathway (Dev et al, 2018; Findlay et al, 2018; Gupta et al, 2018; Noordermeer et al, 2018; Tomida et al, 2018). Some shieldin components have been demonstrated to promote CSR in B-cell lines (Gupta et al, 2018; Noordermeer et al, 2018), and 53BP1/RIF1/REV7-deficient mice have also been observed to have profoundly impaired CSR (Manis et al, 2004; Chapman et al, 2013; Di Virgilio et al, 2013; Ghezraoui et al, 2018). By contrast, the importance of SHLD1, SHLD2, and SHLD3 on CSR in mice has not been tested. In this report, we show that SHLD2 deficiency impairs CSR in mice but has no impact on early B-cell development and V(D)J recombination. We further show that Shld2−/− primary B-cells, as well as 53BP1-, shieldin-, and other NHEJ-deficient CH12F3-2 B-cells (hereafter referred to as CH12 cells), exhibit a significant population with low-to-no expression of Ig upon induction of CSR. This effect is permanent as cells do not recover Ig expression. Our analysis shows that a large proportion of these Iglo CH12 cells have undergone CSR to IgA, however with major deletions of the Ighm and Igha loci that lead to loss of Cα constant region exons. These data suggest that 53BP1 or shieldin deficiency does not lead to reduced recombination at the DNA level per se, but rather to a relative increase in non-productive CSR that inactivates the Igh locus. These results also support a role of the shieldin complex in preventing resection of DNA. Results Lymphocyte development and B-cell populations largely unaffected by Shld2 deficiency Shld2 knockout mice (Shld2em_del/em_del, referred hereafter as Shld2−/−) were generated by microinjection of Cas9 ribonucleoprotein complexes targeting exon 4 in the C57BL/6 background, and a founder mouse with a large out-of-frame deletion (248 bp) was generated (Fig 1A). Histopathology examination of various organs and tissues reported no significant pathologies in the Shld2−/− mice relative to controls (see Materials and Methods). Figure 1. SHLD2 does not affect lymphocyte development or V(D)J recombination A. Schematic of the Shld2 exon 4 showing the 3 sgRNAs used to produce a 251 base pair deletion in one founder line that leads to a frameshift and usage of a premature stop codon. B. Characterization of the various B-cell progenitor fractions as described by Hardy et al (1991) in the bone marrow of 4 wild-type and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Fig EV1A. C. Characterization of the indicated thymic T-cell populations in the bone marrow of 4 wild-type and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Fig EV1D. D. Characterization of the indicated immature and mature splenic B-cell populations in the bone marrow of 4 wild-type and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Fig EV1B. E. Characterization of the indicated peritoneal B-cell populations in the bone marrow of 4 wild-type and 4 Shld2−/− littermate controls. Gating strategy for measuring these populations is shown in Fig EV1C. F. Left panel: Schematic of the A70.2 INV-4 cell line strategy to induce RAG1/2-mediated recombination using imatinib. Right panel: A70.2 INV-4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. Guide RNA targeting chicken AID was used as a negative control (Ctrl). Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured (mean ± SD of 3 biological replicates). The insertion–deletion (indel) penetrance as measured by TIDE analysis (Brinkman et al, 2014) of sequence for each of these sgRNA constructs is shown in Fig EV1E, and the baseline GFP frequency prior to imatinib stimulation is shown in Fig EV1F. sgRNA sequences used are shown in Table EV2. Download figure Download PowerPoint One of the most remarkable genetic interaction observed among DNA repair-coding genes is the ability of 53BP1 mutations to suppress the embryonic lethality and cell lethality associated with BRCA1 loss-of-function alleles (Cao et al, 2009; Bouwman et al, 2010; Bunting et al, 2010). There is currently some debate as to which effector of 53BP1 is responsible to enforce lethality in BRCA1 mutants since the loss PTIP-interacting 53BP1-S25 residue in mice results in viable Brca1Δ11/Δ11 animals suggesting that PTIP is a critical mediator of the embryonic lethality of BRCA1-deficient animals. However, Brca1Δ11/Δ11; 53bp1S25A/S25A mice display phenotypes such as premature aging, and mouse embryo fibroblasts derived from these mice remain profoundly HR-deficient due to the presence of the RIF1-shieldin axis (Callen et al, 2019). To test the impact of shieldin depletion on the viability of the Brca1Δ11 allele (exon 11 deleted), we combined the Shld2-null allele with the Brca1Δ11 mutation by Shld2−/+; Brca1Δ11/+ x Shld2−/+; Brca1Δ11/+ intercrosses. We monitored the genotype of 96 live births and found that introduction of the Shld2 mutation suppressed the embryonic lethality of the Brca1Δ11 allele (Xu et al, 2001) (Table 1; χ2 = 33.208, P = 5.649 × 10−5). At the time of submission, all double-mutant mice were alive and appeared normal, with the oldest mouse being 27.5 weeks old. These results indicate that SHLD2 is a key effector of 53BP1 with respect to the lethality of BRCA1 loss. Table 1. Genotypes of the Shld2+/−; Brca1Δ11/+ x Shld2+/−; Brca1Δ11/+ intercross Genotype Total mice Observed frequency Expected frequency Shld2+/+; Brca1+/+ 8 0.0833 0.0625 Shld2+/−; Brca1+/+ 6 0.0625 0.125 Shld2−/−; Brca1+/+ 10 0.1042 0.0625 Shld2+/+; Brca1Δ11/+ 12 0.1250 0.125 Shld2+/−; Brca1Δ11/+ 37 0.3854 0.25 Shld2−/−; Brca1Δ11/+ 14 0.1458 0.125 Shld2+/+; Brca1Δ11/Δ11 0 – 0.0625 Shld2+/−; Brca1Δ11/Δ11 0 – 0.125 Shld2−/−; Brca1Δ11/Δ11 9 0.0938 0.0625 Total 96 χ2 = 33.208, P = 5.649 × 10−5. In Shld2−/− mice, there was no apparent block in B- and T-cell development in the bone marrow and thymus, respectively, particularly at the Hardy fraction C or DN3 populations corresponding to the pre-BCR and pre-TCR selection stages (Figs 1B and C, and EV1A and D). Moreover, marginal zone and follicular B-cells in the spleen, as well as B1 cells in the peritoneal cavity, were unaffected by SHLD2 deficiency (Figs 1D and E, and EV1B and C). This apparent lack of a defect in lymphocyte developmental suggests that V(D)J recombination mediated by the RAG1/2 recombinase is unaffected by SHLD2 deficiency. To test this notion, we transduced A70.2 INV-4 cell line with CRISPR lentivirus targeting the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. In this cell line, imatinib-induced RAG-mediated recombination of a genomically integrated artificial substrate results in GFP expression (Bredemeyer et al, 2006). In the bulk-edited A70.2 cells, despite similar indel penetrance using the various CRISPR constructs and low baseline frequency of GFP expression (Fig EV1E and F), the only defect in GFP expression was in cells transduced with Lig4-targeting sgRNA (Fig 1F). Hence, SHLD2 deficiency does not impact V(D)J recombination, consistent with what was observed in the Mb1cre/+ Rev7fl/fl mice (Ghezraoui et al, 2018). Click here to expand this figure. Figure EV1. Gating strategies for the assessment of B- and T-cell populations in WT and Shld2−/− mice A. Gating strategies used to quantitate Hardy fractions A-F of progenitor B-cells in bone marrows of WT and Shld2−/− mice. B. Gating strategies used to quantitate immature, mature, T1, T2, MZ, and FO B-cell populations in the spleen of WT and Shld2−/− mice. C. Gating strategies used to quantitate B1, B2, B1a, and B1b populations in the peritoneal cavity of WT and Shld2−/− mice. D. Gating strategies used to quantitate various thymocyte populations in WT and Shld2−/− mice. E. Insertion–deletion (indel) penetrance was measured by TIDE sequencing for the lentiCRISPRv2 constructs expressing the indicated sgRNAs targeting the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. F. Baseline GFP frequency of bulk gene-edited A70.2 cells prior to imatinib stimulation (Fig 1F). Download figure Download PowerPoint SHLD2 is necessary for class switch recombination To determine whether SHLD2 functions in CSR in vivo, we first examined the steady-state levels of serum Ig isotypes. We found that the levels of IgM in the serum of unimmunized Shld2−/− mice were normal, but IgG2b and IgG3 isotypes had reduced concentrations relative to wild type (Fig 2A). Interestingly, IgA levels seemed to be elevated in Shld2−/− animals, perhaps pointing to a reduced dependence of IgA CSR on NHEJ, as previously reported (Li et al, 2018). To further test the role of SHLD2 in CSR, we purified splenic B-cells and induced them to switch to various isotypes using different stimulation cocktails. We found that ex vivo CSR to all tested Ig isotypes shows a clear impairment of Shld2−/− B-cells relative to WT (Figs 2B and EV2A), and this impairment was not due to a decrease in AID protein or sterile transcript expression (Fig EV2B and C). In addition, using an experimental system in which class switch is mediated by Cas9 (Ling et al, 2018), we found that neither 53BP1 nor SHLD2 deficiency affected this pseudo-CSR (Fig EV2D). This finding is similar to previous results involving 53BP1 deficiency and I-SceI-mediated CSR (Bothmer et al, 2010), pointing to a unique relationship between 53BP1/shieldin function and AID-mediated DNA damage. Figure 2. SHLD2-deficient mice have defects in class switch recombination A. Concentration of the various indicated isotypes in the serum of 6- to 8-week-old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; **P ≤ 0.01, unpaired two-tailed t-test. B. B-cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails (Li et al, 2018). Cells were then analyzed by flow cytometry for expression of the various indicated isotypes, and the percent expression of each isotype is reported. Values are mean frequency ± SD of 4 biological replicates; ****P ≤ 0.0001, unpaired two-tailed t-test. C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, the serum was withdrawn 2 weeks post-immunization, and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates. D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated, and anti-IgG-secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2–/– (n = 3); *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Sterile transcript or AID protein levels were unaffected in Shld2−/− B-cells A. Representative flow plots of switched ex vivo B-cells in Fig 2B. B. Purified splenic B-cells from WT and Shld2−/− mice were unstimulated or stimulated for 2 days with LPS + IL4, and germline (sterile) transcripts for Iμ (left panel) and Iγ1 (right panel) were quantitated by qPCR and compared to HPRT mRNA levels. Data are shown relative to WT, which is set at 1. C. Lysates of purified splenic B-cells from WT and Shld2−/− mice that were stimulated for 3 days with LPS + IL4 and subjected to Western blot analyses for AID and the internal control β-actin. D. Cas9-induced switching was carried out on Aid−/−, Aid−/− Lig4−/−, Aid−/− 53bp1−/−, and Aid−/− Shld2−/−/− CH12 clones and switching to IgA was measured 3 days post-transfection. The percent of Iglo cells is also reported. Transfection with the empty vector pX330 served as negative control. Values are mean frequency ± SD of 3 biological replicates; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. Download figure Download PowerPoint To examine the effect of SHLD2 on antigen-specific CSR, mice were immunized with 4-Hydroxy-3-nitrophenylacetyl Chicken Gamma Globulin (NP-CGG). Shld2−/− mice had reduced NP-specific serum of the IgG1, IgG2a, IgG2b, and IgG3 classes relative to WT, as well as a trend to increased NP-specific IgM (Fig 2C). In some cases, the NP-specific serum Ig defect in Shld2−/− mice was intermediate between WT and 53bp1−/− animals. In addition, Shld2−/− splenic NP-specific IgG1-secreting cells were ~7-fold reduced compared to WT and congruent with 53bp1−/− results (Fig 2D). Moreover, there was no apparent difference in splenic germinal center B-cell frequency before or after NP-CGG immunization, suggesting that the Shld2−/− CSR defect is on the molecular level of end joining (Fig EV3). All together, these data show a critical function of SHLD2 in CSR in mice, supporting previous findings in the CH12 B-cell line (Noordermeer et al, 2018). Click here to expand this figure. Figure EV3. Germinal center B-cell frequency is not affected by SHLD2 deficiency A. Germinal B-cell (GL-7+ Fas+) frequency relative to all B-cells (B220+) in the spleen in unimmunized mice; mean ± SD of 3 or 4 biological replicates, ns P ≥ 0.05, unpaired two-tailed t-test. B. As in A, but at day 10 post-NP-CGG immunization; mean ± SD of 4 or 5 biological replicates, ns P ≥ 0.05, unpaired two-tailed t-test. C. As in A, but at day 21 post-NP-CGG immunization; mean ± SD of 3 or 4 biological replicates, ns P ≥ 0.05, unpaired two-tailed t-test. D. Representative flow plots of fluorescence minus one (FMO) controls and day 21 data points. Download figure Download PowerPoint Deficiency in the shieldin complex and other NHEJ factors exhibit a permanent Iglo population upon CSR In carrying out the ex vivo CSR of WT and Shld2−/− B-cells, we observed that Shld2−/− B-cells stimulated with LPS and IL4 showed an increased proportion of IgMlo IgG1lo cells relative to WT cells at day 6 post-stimulation (Fig 3A). This phenomenon was also recapitulated in 53bp1−/−, Shld1−/−, Shld2−/−/−, and Shld3−/− CH12 B-cells at 3 days after stimulating with the CIT cocktail (anti-CD40, IL4, TGFβ) that induces CSR to IgA (Fig 3B). We also found that CH12 cells deficient in other NHEJ factors such as KU70, KU80, DNA-PKcs, XLF, XRCC4, and LIG4 (but not PAXX) (Appendix Fig S1) have reduced CSR to IgA and an increased Iglo population (Fig 3B and C). The presence of this Iglo population was temporary and was reduced by 9 days post-stimulation in ex vivo B-cells (Fig 3A) and 7 days post-CIT stimulation in 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12F3-2 B-cells (Fig 4A), as well as in CH12 cells deficient in Shld1, KU70, KU80, XLF, XRCC4, and LIG4 (Fig EV4A). Figure 3. SHLD2-deficient B-cells and B-cells deficient in other NHEJ factors exhibit an Iglo population upon CSR induction A. WT and Shld2−/− B-cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B-cells 6 days post-stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; **P ≤ 0.01, ***P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test. B. WT CH12 cells, as well as two each of 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient clones generated previously (Noordermeer et al, 2018), as well as a LIG4-deficient CH12 clone, were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry. Representative flow plots for WT and 53bp1−/− CH12 cells are shown at day 3 post-CIT stimulation. The graph plots show proportion of IgA+ and Iglo CH12 cells, mean ± SD from 3 biological replicates; *P ≤ 0.05, ****P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. The letters below the x-axis represent the clone codes for the 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient CH12 clones. One LIG4-deficient CH12 clone was also used. C. The Xlf, Ku70, Ku80, Xrcc4, and Paxx genes were knocked out in CH12 cells by CRISPR, and two independent clones each were analyzed as in Fig 3B with mean ± 3 biological replicates; *P ≤ 0.05, ****P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. Download figure Download PowerPoint Figure 4. Reduced Ig expression in 53BP1-, SHLD2-, and SHLD3-deficient CH12 cells is permanent and dependent on CSR A. WT and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population was reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. B. WT and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post-sort as well as 12 days post-sort (see Fig EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post-CIT treatment is shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. CSR induces a permanent loss of Ig expression in CH12 cells A. The indicated NHEJ-mutant CH12 clones were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression at days 3 and 7; mean ± SD of 3 biological replicates. B. WT, 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 12 days post-sort. Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 12 days of culture post-sort. Download figure Download PowerPoint DSB formation has been shown to transiently reduce expression of a gene, a process mediated by ATM (Harding et al, 2015). Hence, cells deficient in NHEJ factors might have Igh DSBs that persist longer than in WT cells, and thus impose a longer-lasting decrease in Ig expression. To test whether this Iglo population was exhibiting a transient decrease in Ig expression or was being diluted from the population due to increased death and/or decreased proliferation, WT and mutant CH12 cells were stimulated with CIT for 3 days, and IgM+ IgA−, IgM− IgA+, and IgMlo IgAlo populations from WT, 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were sorted out and re-cultured for 5 or 12 days. Notably, all three populations from all CH12 genotypes largely maintained their Ig expression phenotype at the point of sorting (Figs 4B and EV4B). These data suggest that CSR induces a permanent reduced Ig expression in 53bp1−/− and Shldnull CH12 cells. CSR in 53bp1−/− and Shld2−/−/− CH12 cells leads to aberrant recombination involving deletions in the acceptor constant region The finding that CSR induction generates a persistent Iglo population in 53bp1−/− and Shldnull CH12 cells suggests that the Igh locus has been inactivated. One possible explanation for this phenomenon is that 53bp1−/− and other NHEJ-deficient CH12 cells exhibited increased DNA resection during CSR, leading to loss of sequences that allow for surface expression of IgH. To test this notion, Iglo cells from WT, 53bp1−/−, and Shld2−/−/− CH12 cells were sorted, and subcloned, and expression for IgM and IgA was re-assessed by flow cytometry to confirm that Ig expression remained negative or low (Appendix Fig S2). To test whether expression of IgM or IgA was affected at the mRNA level, we used primers that amplified the entire coding region of the IgM and IgA transcript from the leader sequence to stop codon. For the WT Iglo subclones, we found that most expressed IgA at the mRNA level; however, 50% (4/8) of tested subclones only expressed "truncated" (~0.7 kb) IgA cDNAs relative to the full-length IgA mRNA (~1.6 kb); the positive control also has a truncated IgA cDNA band likely corresponding to a mis-spliced product (Fig 5A). For the 53bp1−/− Iglo subclones, 72% (18/25) of tested subclones only expressed truncated IgA cDNAs (~0.7 or 1 kb); a small fraction of subclones expressed neither IgM or IgA cDNA, and at least one clone (D01) appeared oligoclonal by expressing both IgM and IgA transcripts (Fig 5A). Likewise, Shld2−/−/− Iglo subclones also expressed truncated or no IgA transcripts, as compared with Shld2−/−/− IgA+ subclones which expressed the full-length IgA transcript (Fig 5B). Figure 5. Loss of Ig cell-surface expression in CH12 cells is accompanied by aberrant IgA transcripts A. mRNA expression analysis of IgM and IgA was carried out by RT–PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT–PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (b
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