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. 2022 Nov 16;24(1):e55429. doi: 10.15252/embr.202255429

Nuclease‐independent functions of RAG1 direct distinct DNA damage responses in B cells

Rachel Johnston 1, Brendan Mathias 1, Stephanie J Crowley 1, Haley A Schmidt 1, Lynn S White 1, Nima Mosammaparast 2, Abby M Green 1, Jeffrey J Bednarski 1,✉
PMCID: PMC9827558  PMID: 36382770

Abstract

Developing B cells generate DNA double‐stranded breaks (DSBs) to assemble immunoglobulin receptor (Ig) genes necessary for the expression of a mature B cell receptor. These physiologic DSBs are made by the RAG endonuclease, which is comprised of the RAG1 and RAG2 proteins. In pre‐B cells, RAG‐mediated DSBs activate the ATM kinase to coordinate canonical and non‐canonical DNA damage responses (DDR) that trigger DSB repair and B cell developmental signals, respectively. Whether this broad cellular response is distinctive to RAG DSBs is poorly understood. To delineate the factors that direct DDR signaling in B cells, we express a tetracycline‐inducible Cas9 nuclease in Rag1‐deficient pre‐B cells. Both RAG‐ and Cas9‐mediated DSBs at Ig genes activate canonical DDR. In contrast, RAG DSBs, but not Cas9 DSBs, induce the non‐canonical DDR‐dependent developmental program. This unique response to RAG DSBs is, in part, regulated by non‐core regions of RAG1. Thus, B cells trigger distinct cellular responses to RAG DSBs through unique properties of the RAG endonuclease that promotes activation of B cell developmental programs.

Keywords: B cell development, Cas9, DNA breaks, DNA damage response, RAG

Subject Categories: Chromatin, Transcription & Genomics; DNA Replication, Recombination & Repair; Immunology

Both Cas9‐ and RAG‐mediated DNA breaks initiate canonical DNA damage responses in pre‐B cells. RAG‐mediated DNA breaks also uniquely activate non‐canonical developmental signals through properties of the RAG1 endonuclease.

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Introduction

Formation of mature immunocompetent B cells is a highly ordered, multistep process that begins in the bone marrow, where hematopoietic stem cells commit to the B cell lineage and move through discrete stages into secondary lymphoid organs as they build their immunoglobulin (Ig) receptor (Nagasawa, 2006; Melchers, 2015). Each Ig receptor is composed of two immunoglobulin heavy and two immunoglobulin light chains (IgH and IgL, respectively; Rajewsky, 1996). The Igh gene is assembled first in pro‐B cells and pairs with surrogate light chain to generate the pre‐B cell receptor, which subsequently activates Igl gene assembly at either the immunoglobulin kappa (Igk) or lambda (Igλ) locus (Loffert et al, 1996; Herzog et al, 2009). Igh and Igl genes are assembled through the V(D)J recombination reaction, which joins variable (V), diversity (D), and joining (J) gene segments to generate functional exons encoding the antigen‐binding domain (Fugmann et al, 2000a). Ig gene rearrangement occurs in the G1 phase of the cell cycle and requires the generation of DNA double‐stranded breaks (DSBs) at distant DNA segments followed by excision of the intervening DNA sequence and ligation of the intrachromosomal broken DNA ends (Desiderio et al, 1996). The DSBs needed for Ig gene rearrangement are generated by the RAG endonuclease, which is a heterotetramer comprised of two RAG1 and two RAG2 proteins (Schatz et al, 1989; Oettinger et al, 1990; Fugmann et al, 2000a). RAG binds recombination signal sequences (RSSs) at the border of V, D, and/or J segments where it subsequently cleaves DNA. Repair of the DNA ends is mediated by non‐homologous end joining (NHEJ), which introduces diversity into the antigen receptor gene and is critical for preventing errant repair that could lead to translocations (Rooney et al, 2004; Bednarski & Sleckman, 2019).

RAG‐mediated DSBs activate the serine/threonine kinase ATM, which promotes a canonical DNA damage response (cDDR) to coordinate DSB repair (Bredemeyer et al, 2008; Helmink & Sleckman, 2012; Alt et al, 2013; Bednarski & Sleckman, 2019). Through the activity of ATM, DDR proteins, including 53BP1, accumulate at sites of DSBs to protect the open DNA ends and recruit repair factors, which join the broken DNA ends (Anderson et al, 2001; Difilippantonio et al, 2008). In response to RAG DSBs, activation of ATM also induces a pre‐B cell‐specific developmental program (Bredemeyer et al, 2008; Bednarski et al, 2016; Bednarski & Sleckman, 2019). This non‐canonical DNA damage response (ncDDR) includes activation of PIM2 to promote survival of pre‐B cells undergoing Ig rearrangement and induction of transcription factors, including NF‐κB2, which regulate broad gene expression changes (Derudder et al, 2009; Bednarski et al, 2012, 2016). NF‐κB2 induces the transcriptional repressor complex SPIC/BCLAF1, which restricts the proliferation of pre‐B cells and promotes continued B cell maturation (Bednarski et al, 2016; Soodgupta et al, 2019). Collectively, the RAG DSB‐mediated ncDDR supports B cell development while preserving genome integrity.

In pre‐B cells, DNA damage from exogenous sources, such as irradiation, also triggers the conserved cDDR to coordinate repair pathways and cell death programs if the DNA injury is not resolved (Innes et al, 2006, 2013; Bredemeyer et al, 2008; Bednarski & Sleckman, 2019). However, whether these non‐RAG‐mediated DNA breaks also initiate the developmental ncDDR is not yet evident. Prior work has delineated some similarities in transcriptional responses to irradiation‐induced DNA injury and RAG DSBs in pre‐B cells (Bredemeyer et al, 2008; Innes et al, 2013, 2020). These findings suggest that pre‐B cells are inherently poised to initiate both cDRR and ncDDR to any DSBs. However, distinctions in gene expression triggered by these two modes of DNA injury raise the question of whether specific cellular and molecular mechanisms may modulate responses to DSBs to effect differential fates for cells undergoing programmed RAG‐mediated Ig rearrangement versus those with unintended, non‐RAG DNA injury.

In addition to its function in V(D)J recombination, the RAG complex interacts with a diverse protein network that regulates protein localization, protein stability, and DSB formation and repair (Elkin et al, 2005; Matthews et al, 2007; Jones & Simkus, 2009; Coster et al, 2012; Kassmeier et al, 2012; Lescale & Deriano, 2017; Brecht et al, 2020). RAG1 harbors the primary DNA‐binding and cleavage activity while RAG2 functions as an accessory factor enforcing chromatin association and localization (Helmink & Sleckman, 2012; Alt et al, 2013). The endonuclease activity of RAG1 resides in its core domain (amino acids 384–1008), which is essential for RAG complex localization to RSSs and DSB generation (Sadofsky et al, 1993; Silver et al, 1993). The N‐terminal region (NTR; amino acids 1–383) and the C‐terminal tail (amino acids 1009–1040) of RAG1, defined as the non‐core regions of the protein, have critical functions in regulating its activity through association with interacting proteins (Grundy et al, 2010; Kim et al, 2015). RAG1 NTR interacts with MDC1, SF3A2, VprBP, and nucleolar proteins and also contains an E3 ubiquitin ligase domain that regulates accessory factor activities (Simkus et al, 2009; Coster et al, 2012; Kassmeier et al, 2012; Deng et al, 2015; Brecht et al, 2020). The importance of these non‐core regions of RAG1 in lymphocyte development is underscored by the identification of mutations in these domains in patients with primary immune deficiency (Lee et al, 2014; Notarangelo et al, 2016). Further, mice expressing only core RAG1 exhibit aberrant V(D)J recombination, abnormal lymphocyte development, and increased incidence of malignancies, suggesting a key role of these regions in RAG function and immune development (Talukder et al, 2004; Deriano et al, 2011; Horowitz & Bassing, 2014; Beilinson et al, 2021).

Here, we utilize an inducible Cas9 nuclease to elucidate mechanisms of ncDDR activation in response to DSBs in pre‐B cells. We find that both Cas9‐ and RAG‐mediated DSBs initiate cDDR but only RAG DSBs trigger the developmental ncDDR pathways. This distinct response to RAG DSBs is mediated by non‐core domains of RAG1 in a manner independent of its E3 ligase activity. Our findings establish that RAG1 has critical functions in regulating DNA damage signaling in pre‐B cells undergoing Ig recombination.

Results and Discussion

RAG DSBs at Ig alleles activate cDDR and ncDDR in pre‐B cells

To elucidate the factors that coordinate ncDDR programs in pre‐B cells in response to RAG DSBs, we used Rag1 −/− :Bcl2 and Art −/− :Bcl2 Abelson kinase transformed pre‐B cells (abl pre‐B cells). Expression of the Abl kinase promotes cell proliferation and suppresses Rag1 and Rag2 expression. Inhibition of the Abl kinase with imatinib induces G1 arrest, induction of RAG, and recombination of Igk (Bredemeyer et al, 2008). Expression of the Bcl2 transgene in abl pre‐B cells supports the survival of imatinib‐treated, G1‐arrested pre‐B cells, which permits evaluation of DDR signaling. Rag1 −/− :Bcl2 abl pre‐B cells do not generate RAG DSBs and, thus, do not activate DDR signals (Fig 1A). In contrast, since Artemis is required for DSB repair, Art −/− :Bcl2 abl pre‐B cells generate persistent RAG DSBs at Igk, which activate DDR (Rooney et al, 2003; Bredemeyer et al, 2008; Soodgupta et al, 2019). Prior to imatinib treatment, Rag1 −/− :Bcl2 and Art −/− :Bcl2 abl pre‐B cells are cycling and do not have DSBs (Appendix Fig S1A and B). Following the addition of imatinib, both Rag1 −/− :Bcl2 and Art −/− :Bcl2 abl pre‐B cells are arrested in G1 (Appendix Fig S1A). In contrast to Rag1‐deficient pre‐B cells, Art −/− :Bcl2 abl pre‐B cells generated RAG DSBs in ~50% of Igk loci and demonstrated accumulation of the DDR factor 53BP1 in 1–2 foci per cell (Figs 1A and B, and EV1A), indicative of RAG DSBs at Igk alleles. Additionally, pre‐B cells with RAG DSBs had increased phosphorylation of the chromatin modulator KAP1 (TRIM28) without changes in total KAP1 (Figs 1C, and EV1B and C). Both 53BP1 foci and KAP1 phosphorylation are hallmarks of cDDR signaling (Anderson et al, 2001; Difilippantonio et al, 2008; Tubbs et al, 2014). RAG DSBs at Igk also triggered B cell‐specific ncDDR as evidenced by activation of the NF‐κB2 transcription factor (i.e., processing of the p100 precursor to the transcriptionally active p52), induction of Cd40 and Pim2 mRNA transcripts, and expression of PIM2 protein in Art −/− :Bcl2 abl pre‐B cells but not Rag1 −/− :Bcl2 abl pre‐B cells (Figs 1C and D, and EV1B). These findings are consistent with previous studies and demonstrate that RAG‐mediated DSBs at Igk activate both canonical and non‐canonical DDR (Bredemeyer et al, 2008; Bednarski et al, 2012, 2016).

Figure 1. RAG DSBs at Igk and Igh activate cDDR and ncDDR.

Figure 1

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  • A–D
    Rag1 −/− :Bcl2 and Art −/− :Bcl2 abl pre‐B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut), and primer location. Results are normalized to Rag1 −/− :Bcl2 abl pre‐B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation. (B) Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (C) Western blot analysis of KAP1 phosphorylation (p‐KAP1), NF‐κB2 (p100 and p52), and PIM2. GAPDH is shown as a loading control. Quantitation in Fig EV1B. (D) Cd40 and Pim2 mRNA expression.
  • E–H
    Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were transduced with an empty vector (control) or vector expressing RAG1, then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (J H 1) genomic DNA and analyzed as in (A) with results normalized to empty vector control, which has only germline Igh DNA. (F) Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (G) Western blot analysis of FLAG‐RAG1, p‐KAP1, NF‐κB2, and PIM2. GAPDH is shown as a loading control. Quantitation in Fig EV1D. (H) Cd40 and Pim2 mRNA expression.

Data information: In (A, B, D, E, F, and H) data are mean ± SEM for three biological replicates. In (C and G) data are representative of three biological replicates. Significance by Student's t‐test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Source data are available online for this figure.

Figure EV1. RAG DSBs are selectively induced at specific Ig loci depending on pre‐B cell genotype.

Figure EV1

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  1. Distribution of foci per cell in Fig 1B.
  2. Quantitation of protein changes in Western blots for Fig 1C.
  3. Western blot analysis of total KAP1. GAPDH is shown as a loading control. The bar graph shows the quantitation of protein changes.
  4. Germline Igh (J H 1) locus quantified by qPCR analysis in Rag1 −/− :Bcl2 and Art −/− :Bcl2 abl pre‐B cells prior to imatinib treatment. Schematic shows germline (GL) Igh locus, unrepaired JH1 coding end (Cut), representative joined Igh locus (Joined), and primer location. Results are normalized to Rag1 −/− :Bcl2 abl pre‐B cells, which have germline Igh DNA. The absence of Igh germline product in Art −/− :Bcl2 abl pre‐B cells indicates the locus has already rearranged prior to imatinib treatment and induction of RAG. Art −/− :Bcl2 abl pre‐B cells had no PCR product for germline Igh (absence of column and associated error bars).
  5. Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were retrovirally transduced with empty vector (control) or vector expressing RAG1 and then treated with imatinib for 48 h as in Fig 1E. DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA as in Fig 1A.
  6. Distribution of foci per cell in Fig 1F.
  7. Quantitation of protein changes in Western blots for Fig 1G.
  8. Western blot analysis of total KAP1 for cells treated as in Fig 1G. GAPDH is shown as a loading control. The bar graph shows the quantitation of protein changes.

Data information: In (A, B, and D–G) bar graphs are mean ± SEM for three biological replicates. In (C and H) images and bar graphs are for two biological replicates. Significance by Student's t‐test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Source data are available online for this figure.

To determine if RAG DSBs at other Ig genes induce similar DDR in pre‐B cells, we generated Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells. Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells do not generate RAG DSBs and are unable to repair DSBs due to the absence of DNA Ligase 4 (LIG4), which is required for ligation of broken DNA ends (Helmink & Sleckman, 2012; Purman et al, 2019). Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells have had no RAG activity and, thus, retain germline Igh (Fig EV1D). In contrast, Igh has already been recombined in Art −/− :Bcl2 abl pre‐B cells (Fig EV1D), which promotes the accessibility of Igk to RAG and subsequent generation of DSBs at Igk (Johnson et al, 2008; Clark et al, 2014). The absence of a recombined Igh in Rag1 −/− :Lig4 −/− :Bcl2 pre‐B cells is expected to direct RAG activity to Igh rather than to Igk. Retroviral expression of FLAG‐tagged RAG1 in Rag1 −/− :Lig4 −/− :Bcl2 pre‐B cells did not induce DSBs in proliferating cells (Appendix Fig S1B); however, following treatment with imatinib (to induce G1 arrest, Appendix Fig S1A, and expression of endogenous RAG2), RAG DSBs were selectively induced at Igh and not at Igk (Figs 1E and EV1E). RAG DSBs were generated at ~50% of Igh loci resulting in 1–2 53BP1 foci per cell (Figs 1E and F, and EV1F), comparable to DSBs at Igk in Art −/− :Bcl2 pre‐B cells (Figs 1A and B, and EV1A). Similar to RAG DSBs at Igk, RAG DSBs at Igh triggered phosphorylation of KAP1 as well as activation of NF‐κB2, increased PIM2 protein, and induction of Cd40 and Pim2 transcripts (Figs 1G and H, and EV1G and H). Thus, RAG‐mediated DSBs at either Igh or Igk similarly induce both cDDR and ncDDR signaling in early B cells.

ncDDR is not activated by non‐RAG, non‐Ig DSBs in pre‐B cells

The similar DDR programs activated by RAG DSBs at Igh and Igk raised the question of whether all DSBs in pre‐B cells trigger identical cellular responses. Previous work has shown that irradiation‐induced DNA damage results in some of the same cellular responses as RAG DSBs, suggesting that pre‐B cells may have a generalized response to any DNA insult (Bredemeyer et al, 2008; Innes et al, 2013, 2020). However, there are challenges in comparing irradiation‐induced DSBs to RAG DSBs. Irradiation generates DNA breaks, both double‐stranded and single‐stranded, randomly throughout the genome and concomitantly activates other signals, such as reactive oxygen species. In contrast, RAG DSBs occur at targeted locations and do not induce these other signals. To circumvent these confounding factors, we transduced Rag1 −/− :Lig4 −/− :Bcl2 pre‐B cells with a lentivirus to stably express a tetracycline‐inducible FLAG‐tagged Cas9 (Rag1 −/− :Lig4 −/− :Bcl2:iCas9; Purman et al, 2019). Treatment with doxycycline and imatinib induced Cas9 expression and G1 arrest, respectively (Fig 2A and Appendix Fig S1A). Subsequent transfection with a guide RNA (gRNA) promotes Cas9‐mediated DSBs at a targeted locus; thereby, permitting the inducible generation of non‐RAG DSBs at any genomic location (Mali et al, 2013; Sternberg et al, 2014; Purman et al, 2019). Of note, Cas9, similar to the RAG complex, remains bound to cut DNA ends (Agrawal & Schatz, 1997; Arnal et al, 2010; Wang et al, 2012; Sternberg et al, 2014; Brinkman et al, 2018). To determine if pre‐B cells activate a universal DDR to all DSBs, we independently transfected Rag1 −/− :Lig4 −/− :Bcl2:iCas9 with gRNAs targeting the T cell receptor α chain gene enhancer (Eb) or Gapdh. Cas9‐mediated DSBs were generated at both targeted loci as demonstrated by the loss of germline DNA PCR product (Fig 2B and C). DSBs did not occur at putative off‐target sites despite persistent expression of Cas9 and gRNAs, which demonstrates the specificity of this approach in generating targeted DSBs (Fig EV2A and B). To compare DDR signals activated by Cas9 DSBs and RAG DSBs, cellular responses were measured 48 h after initiation of DSBs. As expected, Cas9 DSBs activated cDDR as evidenced by the accumulation of 53BP1 foci and phosphorylation of KAP1 in cells with targeted gRNA compared to those with no gRNA (Figs 2D and E, and EV2C–E). 53BP1 was present in 1–2 foci per cell consistent with DSBs occurring only at targeted alleles and not diffusely throughout the genome (Figs 2D and EV2C). Induction of 53BP1 foci by Cas9 DSBs was similar to responses triggered by RAG DSBs (compare Figs 2D and EV2C to Figs 1F and EV1F). Cas9 DSBs induced phosphorylation of KAP1 (Figs 2E and EV2D) but to a lesser extent than triggered by RAG DSBs (Figs 1G and EV1G). This difference suggests the genomic location of DSBs may impact the magnitude of cDDR signaling in pre‐B cells. In regard to ncDDR, Cas9‐mediated DSBs at Eb and Gapdh did not trigger activation of NF‐κB2, increased expression of PIM2, or induction of Cd40 or Pim2 mRNA expression compared to cells without gRNA or cells with RAG DSBs (Figs 2E and F, and EV2D). Thus, Cas9‐mediated DSBs only activated cDDR programs but not ncDDR signaling. These findings reveal that ncDDR signals are not a conserved component of pre‐B cell responses to all DSBs.

Figure 2. Cas9 DSBs at non‐Ig genes do not activate ncDDR.

Figure 2

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  • A
    Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated with (+) or without (−) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG‐Cas9 and GAPDH (loading control).
  • B–F
    Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated with doxycycline as in (A) and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs was quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (−). Schematic shows germline (GL) locus, unrepaired cut end (Cut), and location of gRNA and primers. Loss of germline product is representative of DSB generation. (D) Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (E) Western blot analysis of FLAG‐tagged Cas9 or RAG1 (as indicated), p‐KAP1, NF‐κB2, and PIM2. GAPDH is shown as a loading control. Responses to RAG DSBs in Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison. # Denotes band for FLAG‐Cas9. * Denotes band for FLAG‐RAG1. Quantitation in Fig EV2D. (F) Cd40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.

Data information: In (B, C, D, and F) data are mean ± SEM for three biological replicates. In (A and E) data are representative of three biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05.

Source data are available online for this figure.

Figure EV2. Off‐target activity and quantitation of DDR in pre‐B cells with Cas9 DSBs at non‐Ig loci.

Figure EV2

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Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated as in Fig 2. All analyses were completed 48 h after gRNA transfection.
  • A, B
    Schematics show putative off‐target binding sites of indicated gRNAs (red asterisk), germline (GL) locus of putative off‐target sites, and location of PCR primers. Germline locus was quantified by qPCR analysis across Cdkn2aip (A) and Cdhr3 (B).
  • C
    Distribution of foci per cell in Fig 2D.
  • D
    Quantitation of protein changes in Western blots for Fig 2E.
  • E
    Western blot analysis of total KAP1 for cells treated as in Fig 1G. GAPDH is shown as a loading control. The bar graph shows the quantitation of protein changes.

Data information: In (A–D) bar graphs are mean ± SEM for three biological replicates. In (E) images and bar graphs are for two biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05.

Source data are available online for this figure.

Cas9 DSBs at Ig alleles activate cDDR but not ncDDR in pre‐B cells

Activation of ncDDR may be regulated by unique features of the RAG endonuclease or specific characteristics of Ig loci. To resolve this, Rag1 −/− :Lig4 −/− :Bcl2:iCas9 were treated with imatinib and doxycycline to induce cell cycle arrest and Cas9 expression, respectively, then independently transfected with distinct gRNAs targeting the RSS of the Jκ1, J H 1, or D H 1‐1 gene segment. All three gRNAs were designed to generate a Cas9 DSB at or very near the location where a RAG DSB would be generated. Cas9 DSBs were generated at each antigen receptor allele to an equivalent extent as observed with expression of RAG (Fig 3A and B; compared to Fig 1E). As above, Cas9 DSBs did not occur at putative off‐target sites (Fig EV3A–C). Cas9‐mediated DSBs at both Igk and Igh activated cDDR as evidenced by the generation of 53BP1 foci and phosphorylation of KAP1 (Figs 3C and D, and EV3D–F). Again, cells generated 1–2 53BP1 foci reflective of DSBs occurring selectively at the targeted alleles (Figs 3C and EV3D). However, Cas9 DSBs at neither Igk nor Igh triggered ncDDR as they did not induce NF‐κB2 activation or expression of CD40 and Pim2 compared to cells without gRNA (Figs 3D and E, and EV3E).

Figure 3. Cas9 DSBs at Ig genes do not activate ncDDR.

Figure 3

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Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated with imatinib and doxycycline as in Fig 2, then transfected with Jκ1, J H 1, J H D1‐1, or both J H 1 + J H D1‐1 gRNAs as indicated. Following transfection, cells were maintained in doxycycline and imatinib with all analyses completed 48 h after gRNA transfection.
  • A, B
    DSBs quantified by qPCR analysis of Jκ1 (A) and J H 1 and J H D1‐1 (B) genomic DNA. Schematics show germline (GL) locus and location of respective gRNAs and primers. PCR is normalized to cells without gRNA (−). Loss of germline product is representative of DSB generation.
  • C
    Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.
  • D
    Western blot analysis of FLAG‐Cas9, p‐KAP1, NF‐κB2, and PIM2. GAPDH is shown as a loading control. Quantitation in Fig EV3E.
  • E
    CD40 and Pim2 mRNA expression. The Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.

Data information: In (A, B, C, and E) data are mean ± SE for three biological replicates. In (D) data are representative of three biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01.

Source data are available online for this figure.

Figure EV3. Off‐target activity and quantitation of DDR in pre‐B cells with Cas9 DSBs at Ig loci.

Figure EV3

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Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated as in Fig 3. All analyses were completed 48 h after gRNA transfection.
  • A–C
    Schematics show putative off‐target binding sites of indicated gRNAs (red asterisk), germline (GL) locus of putative off‐target sites, and location of PCR primers. Germline locus was quantified by qPCR analysis across Sxn19 (A), Gab1 (B), and Pou6f2 (C). Results are normalized to cells without gRNA (−).
  • D
    Distribution of foci per cell in Fig 3C.
  • E
    Quantitation of protein changes in Western blots for Fig 3D.
  • F
    Western blot analysis of total KAP1 for cells treated as in Fig 3D. GAPDH is shown as a loading control. The bar graph shows the quantitation of protein changes.

Data information: In (A–E) bar graphs are mean ± SEM for three biological replicates. In (F) images and bar graphs are for two biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05, ***P ≤ 0.001.

Source data are available online for this figure.

A key difference between RAG‐ and Cas9‐mediated breaks is the number of DSBs generated. RAG generates two simultaneous DSBs. In Igk, DSBs are generated in the V and J gene segments (Fugmann et al, 2000a). During Igh recombination, RAG first creates DSBs in the D and J gene segments and then in the V and DJ gene segments (Fugmann et al, 2000a). In contrast, Cas9 generates a single DSB in the target allele per gRNA. To determine if the multiplicity of DSBs impacts DDR signaling, Rag1 −/− :Lig4 −/− :Bcl2:iCas9 pre‐B cells were transfected with two gRNAs to simultaneously generate DSBs at J H 1 and D H 1‐1 to more accurately approximate RAG DSBs (Fig 3B). Similar to results with a single Cas9 DSB, generation of paired Cas9 DSBs in Igh activated cDDR but not ncDDR (Figs 3C–E and EV3D–F). Importantly, the absence of ncDDR activation is not secondary to the effects of nucleofection as mock transfection of Rag1 −/− :Lig4 −/− :Bcl2 pre‐B cells expressing FLAG‐RAG1 did not alter the generation of RAG DSBs or activation of either cDDR or ncDDR (Fig EV4A–E). In sum, these results demonstrate that Cas9 and RAG DSBs at Ig alleles activate distinct DDR signals. While both Cas9 and RAG DSBs activate cDDR, the ncDDR program is uniquely initiated by RAG DSBs (compare Figs 1 and 3). Given that Cas9 and RAG DSBs were generated at the same genomic location, these findings suggest that unique features of the RAG endonuclease itself are responsible for coordinating the ncDDR response.

Figure EV4. Nucleofection does not impair activation of ncDDR by RAG DSBs.

Figure EV4

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Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were transduced with an empty vector (control) or vector expressing RAG1 as in Fig 1E. Cells were treated with imatinib for 24 h then nucleofected (no gRNA) and maintained in imatinib as in Figs 2 and 3. Analyses were done 24 h after nucleofection (48 h after imatinib and DSB initiation).
  1. DSBs quantified by qPCR analysis of Igh (J H 1) genomic DNA as in Fig 1E.
  2. Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.
  3. Distribution of foci per cell in panel (B).
  4. Western blot analysis of FLAG‐RAG1, p‐KAP1, NF‐κB2, and PIM2. GAPDH is shown as a loading control.
  5. Cd40 and Pim2 mRNA expression.

Data information: In (A–C and E) data are mean ± SEM for three biological replicates. In (D) data are representative of three biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Source data are available online for this figure.

RAG1 non‐core domains are critical for the activation of ncDDR

The non‐core domains of RAG1, particularly the NTR, have been shown to regulate RAG complex stability, localization, and recombination activity (Elkin et al, 2005; Matthews et al, 2007; Jones & Simkus, 2009; Coster et al, 2012; Kassmeier et al, 2012; Lescale & Deriano, 2017; Brecht et al, 2020). To test whether these regions of RAG1 function in DDR signaling, we retrovirally transduced Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells with the following RAG1 variants: (i) full‐length RAG1, (ii) core RAG1 (cRAG1, C), which lacks the entire N‐terminal and C‐terminal regions but retains the endonuclease domain, (iii) RAG1 with inactivating P326G mutation of the E3 ligase catalytic site (RAG1P326G, PG), and (iv) endonuclease‐inactive RAG1 with D708A mutation within its catalytic domain (RAG1D708A, DA; Fig 4A; Kim et al, 1999; Fugmann et al, 2000b; Dudley et al, 2003; Yurchenko et al, 2003; Simkus et al, 2007; Ji et al, 2010; Beilinson et al, 2021). Cells were subsequently treated with imatinib to induce G1 arrest and expression of endogenous RAG2. cRAG1 and RAG1P326G were previously shown to have diminished recombinase activity, reduced repair of RAG DSBs, and impaired Igk recombination compared to full‐length RAG1 (Dudley et al, 2003; Simkus et al, 2007; Grazini et al, 2010; Beilinson et al, 2021). Using a repair‐deficient (Lig4 −/−) background, we centered our investigations on DDR signaling in response to the generated RAG DSBs independent of effects from alterations in DSB repair. As expected, the catalytically inactive RAG1D708A did not generate DSBs and, accordingly, neither cDDR nor ncDDR programs were activated (Figs 4B–E and EV5A–E). Expression of cRAG1, RAG1P326G, and full‐length RAG1 in Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells resulted in the generation of equivalent levels of DSBs in Igh (Fig 4B). Further, pre‐B cells expressing full‐length RAG1, cRAG1, or RAG1P326G accumulated similar numbers and distribution of 53BP1 foci at both 24 and 48 h after imatinib treatment consistent with comparable magnitude and kinetics of DSB generation (Figs 4C and EV5A–C). Additionally, KAP1 phosphorylation was equivalent for all three RAG1 variants (Figs 4D, and EV5D and E). Thus, DSBs generated by cRAG1 or RAG1P326G activate cDDR (i.e., 53BP1 foci and p‐KAP1) similarly to cells expressing full‐length RAG1.

Figure 4. Non‐core regions of RAG1 are necessary for the activation of ncDDR.

Figure 4

Open in a new tab
Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were retrovirally transduced with empty vector or vector expressing indicated RAG1 variants and then treated with imatinib for 48 h.
  1. Schematic of RAG1 variants. Figure not to scale.
  2. DSBs quantified by qPCR analysis of Igh (J H 1) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control, which has only germline Igh DNA. There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full‐length RAG1.
  3. Representative images of 53BP1 foci. The scale bar denotes 8 μm. The bar graph shows the percentage of cells with the indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.
  4. Western blot analysis of FLAG‐RAG1, p‐KAP1, NF‐κB2, and PIM2. GAPDH is shown as a loading control. Quantitation in Fig EV5D.
  5. Cd40 and Pim2 mRNA expression.

Data information: In (B, C, and E) data are mean ± SEM for three biological replicates. In (D) data are representative of three biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Source data are available online for this figure.

Figure EV5. Quantitation of DDR triggered by RAG1 variants.

Figure EV5

Open in a new tab
Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were retrovirally transduced with empty vector or vector expressing indicated RAG1 variants and treated as in Fig 4.
  1. Representative images of 53BP1 foci at 24 h after imatinib treatment. The scale bar denotes 8 μm.
  2. Quantitation of foci per cell in (A). The bar graph shows the percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.
  3. Distribution of foci per cell in Fig 4C (at 48 h after imatinib).
  4. Quantitation of protein changes in Western blots for Fig 4D.
  5. Western blot analysis of total KAP1 for cells treated as in Fig 4D. GAPDH is shown as a loading control. The bar graph shows the quantitation of protein changes.

Data information: In (A) images are representative of three biological replicates. In (B–D) data are mean ± SEM for three biological replicates. In (E) images and bar graphs are for two biological replicates. Significance by Student's t‐test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Source data are available online for this figure.

Despite comparable DSBs and cDDR signaling, expression of cRAG1, in contrast to full‐length RAG1 and RAG1P326G, did not activate NF‐κB2 or trigger increased PIM2 protein (Figs 4D and EV5D). However, cRAG1‐mediated DSBs did induce Cd40 and Pim2 mRNA transcripts similar to DSBs generated by RAG1 and RAG1P326G (Fig 4E). Thus, cRAG1‐mediated DSBs activate some but not all components of ncDDR signaling. Collectively, these results demonstrate that induction of the ncDDR depends upon the generation of a DSB by RAG and that elements of this developmental program are coordinated by the non‐core regions of RAG1, independent of its E3 ligase activity.

The differential cellular responses to Cas9‐ versus RAG‐mediated DSBs reveal that pre‐B cells trigger distinct DDR signals based on the mechanism of DNA injury. While all DSBs activate canonical pathways to promote DSB repair, RAG DSBs uniquely trigger a developmental program that regulates pre‐B cell survival and differentiation. PIM2 supports the survival of pre‐B cells with RAG DSBs to permit time for the completion of Ig recombination and multiple rearrangements necessary for antigen receptor diversification (Derudder et al, 2009; Bednarski et al, 2012). In pre‐B cells with RAG DSBs, NF‐κB2 drives expression of the SPIC/BCLAF1 transcriptional repressor complex, which modulates PU.1 activity to regulate a broad cellular program, including suppression of SYK and pre‐BCR signaling (Bednarski et al, 2016; Soodgupta et al, 2019). Selective activation of these programs by RAG DSBs is likely beneficial to developing pre‐B cells as it promotes the maturation of cells with recombined Ig loci. In contrast, this process is not triggered in cells with non‐RAG‐mediated DSBs where initiation of the developmental DDR program could result in aberrant maturation of B cells that do not express an appropriately rearranged Ig, which poses risks for the development of autoreactive clones or leukemic transformation.

In addition to the contributions of RAG DSBs, locus‐specific determinants may also influence DDR signaling in pre‐B cells. In this respect, Cas9 DSBs at non‐Ig loci induced a low level of KAP1 phosphorylation in contrast to the higher levels stimulated by both Cas9 DSBs and RAG DSBs at Igh. All DSBs, regardless of location or mechanism, generated 53BP1 foci to a similar extent. Thus, cDDR is activated by all DSBs in pre‐B cells, but the magnitude of downstream signaling may be impacted by the genomic location of the DNA injury with more robust responses triggered by DSBs in Ig loci. Ig genes have distinct epigenetic and transcriptional characteristics that may promote differential cDDR signaling. It is not evident, though, that differences in the magnitude of cDDR alter cell fate in response to the DSBs.

We show that the unique ncDDR activated by RAG DSBs is regulated, at least in part, by the RAG1 protein itself. Further, our studies identify a novel, critical activity of non‐core regions of RAG1 in coordinating components of the DDR. Induction of Cd40 and Pim2 mRNA occurs through RAG1‐dependent mechanisms that are independent of its non‐core regions. In contrast, the non‐core domains of RAG1 are necessary for the activation of NF‐κB2 and expression of PIM2 protein. Expression of Cd40 and Pim2 mRNA transcripts depends on NF‐κB1 (p50/p65), which is activated by ATM in response to RAG DSBs (Bredemeyer et al, 2008). Our results suggest this component of the ncDDR is intact in the absence of non‐core regions of RAG1 but that post‐transcriptional pathways that promote PIM2 protein expression and activation of NF‐κB2 depend on functions of the non‐core regions of RAG1. We find that loss of RAG1 E3 ligase activity within the NTR does not alter NF‐κB2 activation, expression of Cd40 and Pim2 transcripts, or induction of PIM2 protein. Consistent with our results, Beilinson et al (2021) also demonstrated that RAG1 ubiquitin ligase activity did not regulate Pim2 transcripts or protein in primary pre‐B cells. Thus, RAG1 directs ncDDR in pre‐B cells through both non‐core region‐dependent and ‐independent activities that do not depend on its E3 ligase.

The non‐catalytic domains of RAG1, particularly the NTR, contain highly unstructured or disordered regions that can serve as binding sites for interacting proteins (Jones & Simkus, 2009; Notarangelo et al, 2016). In regards to their activity in DDR signaling, RAG1 non‐core regions may associate with DDR signaling factors to alter their activity or, alternatively, may recruit signaling proteins to sites of DSBs, where they can be modulated by ATM or other DDR kinases to trigger downstream signaling pathways. Additional studies are needed to delineate the precise regions of RAG1 that function in ncDDR signaling. A function of RAG1 non‐core regions in DDR signaling reveals potential mechanisms underlying immune dysregulation in patients with variants in these domains (Lee et al, 2014; Notarangelo et al, 2016). These essential activities of RAG1 ensure that DDR signaling promotes B cell differentiation and diversification while limiting errant B cell development in response to non‐programmed DSBs.

Materials and Methods

Cell lines

Rag1 −/− :Bcl2 and Art −/− :Bcl2 abl pre‐B cells were previously described (Bredemeyer et al, 2008; Soodgupta et al, 2019). Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were generated from Rag1 −/− :Lig4 flox/flox :Bcl2 abl pre‐B cells, which were made from Rag1 −/− :Lig4 flox/flox :Bcl2 mice by retroviral transduction of bone marrow cells with MSCV‐v‐abl plasmid as described previously (Bredemeyer et al, 2006). Rag1 −/− :Lig4 flox/flox :Bcl2 abl pre‐B cells were then transiently transfected with MSCV‐Cre‐IRES‐Thy1.1 vector (Addgene, Plasmid #17442) and subsequently subcloned to identify clones with deletion of Lig4, which was confirmed by genotyping (PCR) and phenotyping (persistent RAG‐mediated DSBs following retroviral expression of RAG1, see below). All cell lines were verified by genotyping and confirmed to be free of mycoplasma. Rag1 −/− :Lig4 −/− :Bcl2 abl pre‐B cells were transduced with lentiviral vector pFLRU‐TRE‐Cas9‐Ubc‐rtTA‐IRES‐Thy1.2 vector to generate Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells with stably integrated inducible Cas9 (Purman et al, 2019). Transduced cells were sorted for Thy1.2 expression and then subcloned. All cell lines were authenticated by genotyping. To induce cell cycle arrest and induction of RAG DSBs, abl pre‐B cells were treated with 3 μM imatinib (106 cells/ml) for indicated times prior to harvesting for genomic DNA, protein, RNA, and immunofluorescence (Bredemeyer et al, 2008; Soodgupta et al, 2019; Johnston et al, 2022). For Cas9 induction, Rag1 −/− :Lig4 −/− :Bcl2:iCas9 abl pre‐B cells were treated with 2 μg/ml doxycycline simultaneously with the addition of imatinib.

Mice

Mice were used for the generation of abl pre‐B cell lines as detailed above. Mice were maintained under specific pathogen‐free conditions at the Washington University School of Medicine and were handled in accordance with the guidelines set forth by the Division of Comparative Medicine of Washington University.

cDNA expression

A pFLRU‐TRE‐Cas9‐Ubc‐rtTA‐Thy1.2 lentiviral vector expressing cDNA encoding 5' FLAG‐tagged Streptococcus pyogene Cas9 was a gift from Gene Oltz and Barry Sleckman (Purman et al, 2019). cDNA encoding 5' FLAG‐tagged RAG1 or cRAG1 was cloned into the pOZ‐IRES‐hCD25 retroviral vector (Bednarski et al, 2016). RAG1 E3 ligase mutant (RAG1P326G) and the catalytically inactive RAG1 (RAG1D708A) were generated using QuikChange II XL (Agilent) according to the manufacturer's protocol. Retrovirus and lentivirus were produced in PlatE cells (Cell Biolabs) and 293T cells, respectively, by transfection of the viral plasmids with Lipofectamine 2000 (Life Technologies; Bednarski et al, 2012; Soodgupta et al, 2019). For lentivirus, pCMV‐VSV‐G and pCMV‐d8.2R were also included in the transfection (Stewart et al, 2003; Soodgupta et al, 2019). Viral supernatant was collected and pooled from 24 to 72 h after transfection. Abl pre‐B cells were transduced with the unconcentrated virus in media with polybrene (5 μg/ml; Sigma‐Aldrich) as previously described (Soodgupta et al, 2019). Transduced cells were identified by flow cytometric assessment of hCD25 or Thy1.2 and were sorted using anti‐hCD25 or anti‐Thy1.2 magnetic beads (Miltenyi Biotec) on MS columns (Miltenyi Biotec) according to the manufacturer's protocol.

Generation of Cas9‐mediated DSBs

gRNAs were designed using CHOPCHOP, with the exception of the Eb guide, which is previously published (Dorsett et al, 2014; Labun et al, 2019; Purman et al, 2019). gRNA sequences are listed in Appendix Table S1. gRNAs were generated by in vitro transcription using the Precision gRNA Synthesis kit (Invitrogen) per manufacturer's guidelines. Rag1 −/− :Lig4 −/− :Bcl2:iCas9 cells were treated with doxycycline and imatinib for 24 h. Subsequently, 20 × 106 cells and 400 μg of gRNA were resuspended in 100 μl Nucleofector Solution for Human B Cells (Lonza) and then nucleofected using an Amaxa Nucleofector II (Lonza), program X‐001, according to the manufacturer's instructions. Nucleofected cells were transferred directly to pre‐equilibrated recovery medium containing doxycycline and imatinib at 10 × 106 cells/ml and incubated for indicated times prior to harvesting for genomic DNA, protein, RNA, and immunofluorescence. Putative off‐target gRNA‐binding sites were identified using Cas‐OFFinder (Bae et al, 2014).

Flow cytometric analyses

All flow cytometric analyses were performed on a Cytek‐modified BD FACScan (BD Biosciences). Antibodies used included phycoerythrin (PE)‐conjugated anti‐hCD25 (Biolegend) and allophycocyanin (APC)‐conjugated anti‐Thy1.2 (Biolegend). Staining with DAPI (4′,6‐diamidino‐2‐phenylindole) was used to quantitate the cell cycle.

Western blot analyses

Western blots were done on whole‐cell lysates as previously described (Bednarski et al, 2016). Anti‐NFκB2 (p100/p52; clone D9S3M) and anti‐GAPDH (clone D16H11) antibodies were from Cell Signaling Technology. Anti‐phospho‐KAP1 antibody (A300‐767A) was from Bethyl Laboratories. The total KAP1 antibody was from Genetex (clone N3C2). Anti‐PIM2 (clone 1D12) was from Santa Cruz Biotechnology. Anti‐FLAG (clone M2) was from Sigma. Secondary reagents were horseradish peroxidase (HRP)‐conjugated anti‐mouse IgG (Cell Signaling) or anti‐rabbit IgG (Cell Signaling). Westerns were developed with ECL (Pierce) and ECL Prime (Cytiva). ImageJ was used to quantitate western blots.

RT–PCR

For genomic DNA isolation, cells were lysed in lysis buffer (100 mM TRIS pH 8.5, 5 mM EDTA, 200 mM NaCl, and 0.2% SDS) and DNA was precipitated by addition of isopropanol, washed with 70% and then 100% ethanol, and finally resuspended in water (Soodgupta et al, 2019; Johnston et al, 2022). For PCR over the break assay, genomic DNA was digested with NEBNext dsDNA Fragmentase (NEB) for 10 min followed by PCR Cleanup (QIAGEN) as per manufacturer's instructions before RT–PCR. RNA was isolated using RNeasy (QIAGEN) and reverse transcribed using a polyT primer with SuperScriptII (Life Technologies) according to the manufacturers' protocol. RT–PCR was performed using Brilliant II SYBR Green (Agilent) and acquired on an MX3000P (Stratagene) or QuantStudio 3 (Thermo Fisher). Each reaction was run in triplicate. For PCR over‐the‐break analyses, values at targeted sites were normalized to PCR product spanning CD19, a control region of uncut genomic DNA, and fold change was determined by the ΔΔ cycle threshold method (Johnston et al, 2022). Primer sequences are listed in Appendix Table S1.

Immunofluorescence

Immunofluorescence microscopy was done as previously described (Brickner et al, 2017). Briefly, cells were applied to coverslips using Cell Tak (Corning) at 37°C for 20 min then extracted with 0.2% Triton in PBS for 1 min on ice and fixed with 3.2% paraformaldehyde. Cells were washed with IF Wash Buffer (PBS, 0.5% NP‐40 and 0.02% NaN3), then blocked with IF Blocking Buffer (10% FBS in IF Wash Buffer) for 30 min at room temperature. Slides were incubated with rabbit anti‐53BP1 (Novus; 1:500) in IF Blocking Buffer for 1 h at room temperature. Slides were washed and then stained with goat anti‐rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen; 1:1,000) and Hoechst 33342 (Sigma‐Aldrich) for 30 min at room temperature followed by sample mounting with Prolong Gold mounting media (Invitrogen). Microscopy was performed on an Olympus fluorescence microscope (BX‐53) using an ApoN 60×/1.49 NA oil immersion lens or an UPlanS‐Apo 100×/1.4 oil immersion lens and cellSens Dimension software. Raw images were exported into Adobe Photoshop, and for any adjustment in image contrast or brightness, the levels' function was applied. Foci were manually counted in triplicate on at least 100 cells for each biological replicate.

Statistical analysis

Statistical analyses were done by Student's t‐test using Prism (GraphPad Software).

Author contributions

Rachel Johnston: Data curation; formal analysis; investigation; methodology; writing – original draft; writing – review and editing. Brendan Mathias: Data curation; formal analysis; investigation; writing – review and editing. Stephanie J Crowley: Data curation; formal analysis; writing – review and editing. Haley A Schmidt: Data curation; formal analysis; writing – review and editing. Lynn S White: Data curation; formal analysis; investigation; writing – review and editing. Nima Mosammaparast: Resources; formal analysis; writing – review and editing. Abby M Green: Resources; formal analysis; writing – review and editing. Jeffrey J Bednarski: Conceptualization; resources; formal analysis; supervision; funding acquisition; methodology; writing – original draft; project administration; writing – review and editing.

Disclosure and competing interests statement

JJB discloses advisory roles for Horizon Therapeutics, Sobi, and Prime Medicine. The authors declare that they have no conflict of interest.

Supporting information

Appendix

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Expanded View Figures PDF

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Source Data for Expanded View

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PDF+

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Source Data for Figure 1

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Source Data for Figure 2

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Source Data for Figure 3

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Source Data for Figure 4

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Acknowledgements

This work was supported by the National Institutes of Health grants K08AI102946 (JJB), R56AI153234 (JJB), R01AI173077 (JJB), R01CA227001 (NM), R01CA193318 (NM), P01CA092584 (NM), and 5T32AI007163 (SJC). JJB was supported by the Foundation for Barnes‐Jewish Hospital Cancer Frontier Fund, Barnard Trust, American Society of Hematology, Gabrielle's Angel Foundation, St. Louis Children's Hospital Foundation, and the Children's Discovery Institute. RJ was supported by a training grant through the Alvin J. Siteman Cancer Center. AMG was supported by the Department of Defense (CA200867) and the Children's Discovery Institute. NM was supported by the Centene Personalized Medicine Initiative, the American Cancer Society (RSG‐18‐156‐01‐DMC), the Barnard Foundation, and the Alvin J. Siteman Cancer Center Investment Program. The visual synopsis was created with BioRender.com.

EMBO Reports (2023) 24: e55429.

Data availability

No primary datasets have been generated or deposited.

References

  1. Agrawal A, Schatz DG (1997) RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 89: 43–53 [DOI] [PubMed] [Google Scholar]
  2. Alt FW, Zhang Y, Meng FL, Guo C, Schwer B (2013) Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell 152: 417–429 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson L, Henderson C, Adachi Y (2001) Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol Cell Biol 21: 1719–1729 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arnal SM, Holub AJ, Salus SS, Roth DB (2010) Non‐consensus heptamer sequences destabilize the RAG post‐cleavage complex, making ends available to alternative DNA repair pathways. Nucleic Acids Res 38: 2944–2954 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bae S, Park J, Kim JS (2014) Cas‐OFFinder: a fast and versatile algorithm that searches for potential off‐target sites of Cas9 RNA‐guided endonucleases. Bioinformatics 30: 1473–1475 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bednarski JJ, Sleckman BP (2019) At the intersection of DNA damage and immune responses. Nat Rev Immunol 19: 231–242 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bednarski JJ, Nickless A, Bhattacharya D, Amin RH, Schlissel MS, Sleckman BP (2012) RAG‐induced DNA double‐strand breaks signal through Pim2 to promote pre‐B cell survival and limit proliferation. J Exp Med 209: 11–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bednarski JJ, Pandey R, Schulte E, White LS, Chen BR, Sandoval GJ, Kohyama M, Haldar M, Nickless A, Trott A et al (2016) RAG‐mediated DNA double‐strand breaks activate a cell type‐specific checkpoint to inhibit pre‐B cell receptor signals. J Exp Med 213: 209–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Beilinson HA, Glynn RA, Yadavalli AD, Xiao J, Corbett E, Saribasak H, Arya R, Miot C, Bhattacharyya A, Jones JM et al (2021) The RAG1 N‐terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination. J Exp Med 218: e20210250 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brecht RM, Liu CC, Beilinson HA, Khitun A, Slavoff SA, Schatz DG (2020) Nucleolar localization of RAG1 modulates V(D)J recombination activity. Proc Natl Acad Sci USA 117: 4300–4309 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bredemeyer AL, Sharma GG, Huang CY, Helmink BA, Walker LM, Khor KC, Nuskey B, Sullivan KE, Pandita TK, Bassing CH et al (2006) ATM stabilizes DNA double‐strand‐break complexes during V(D)J recombination. Nature 442: 466–470 [DOI] [PubMed] [Google Scholar]
  12. Bredemeyer AL, Helmink BA, Innes CL, Calderon B, McGinnis LM, Mahowald GK, Gapud EJ, Walker LM, Collins JB, Weaver BK et al (2008) DNA double‐strand breaks activate a multi‐functional genetic program in developing lymphocytes. Nature 456: 819–823 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brickner JR, Soll JM, Lombardi PM, Vagbo CB, Mudge MC, Oyeniran C, Rabe R, Jackson J, Sullender ME, Blazosky E et al (2017) A ubiquitin‐dependent signalling axis specific for ALKBH‐mediated DNA dealkylation repair. Nature 551: 389–393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Brinkman EK, Chen T, de Haas M, Holland HA, Akhtar W, van Steensel B (2018) Kinetics and Fidelity of the repair of Cas9‐induced double‐Strand DNA breaks. Mol Cell 70: 801–813 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Clark MR, Mandal M, Ochiai K, Singh H (2014) Orchestrating B cell lymphopoiesis through interplay of IL‐7 receptor and pre‐B cell receptor signalling. Nat Rev Immunol 14: 69–80 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Coster G, Gold A, Chen D, Schatz DG, Goldberg M (2012) A dual interaction between the DNA damage response protein MDC1 and the RAG1 subunit of the V(D)J recombinase. J Biol Chem 287: 36488–36498 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Deng Z, Liu H, Liu X (2015) RAG1‐mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination. Cell Res 25: 181–192 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Deriano L, Chaumeil J, Coussens M, Multani A, Chou Y, Alekseyenko AV, Chang S, Skok JA, Roth DB (2011) The RAG2 C terminus suppresses genomic instability and lymphomagenesis. Nature 471: 119–123 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Derudder E, Cadera EJ, Vahl JC, Wang J, Fox CJ, Zha S, van Loo G, Pasparakis M, Schlissel MS, Schmidt‐Supprian M et al (2009) Development of immunoglobulin lambda‐chain‐positive B cells, but not editing of immunoglobulin kappa‐chain, depends on NF‐kappaB signals. Nat Immunol 10: 647–654 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Desiderio S, Lin WC, Li Z (1996) The cell cycle and V(D)J recombination. Curr Top Microbiol Immunol 217: 45–59 [DOI] [PubMed] [Google Scholar]
  21. Difilippantonio S, Gapud E, Wong N, Huang CY, Mahowald G, Chen HT, Kruhlak MJ, Callen E, Livak F, Nussenzweig MC et al (2008) 53BP1 facilitates long‐range DNA end‐joining during V(D)J recombination. Nature 456: 529–533 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dorsett Y, Zhou Y, Tubbs AT, Chen BR, Purman C, Lee BS, George R, Bredemeyer AL, Zhao JY, Sodergen E et al (2014) HCoDES reveals chromosomal DNA end structures with single‐nucleotide resolution. Mol Cell 56: 808–818 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Dudley DD, Sekiguchi J, Zhu C, Sadofsky MJ, Whitlow S, DeVido J, Monroe RJ, Bassing CH, Alt FW (2003) Impaired V(D)J recombination and lymphocyte development in core RAG1‐expressing mice. J Exp Med 198: 1439–1450 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Elkin SK, Ivanov D, Ewalt M, Ferguson CG, Hyberts SG, Sun ZY, Prestwich GD, Yuan J, Wagner G, Oettinger MA et al (2005) A PHD finger motif in the C terminus of RAG2 modulates recombination activity. J Biol Chem 280: 28701–28710 [DOI] [PubMed] [Google Scholar]
  25. Fugmann SD, Lee AI, Shockett PE, Villey IJ, Schatz DG (2000a) The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu Rev Immunol 18: 495–527 [DOI] [PubMed] [Google Scholar]
  26. Fugmann SD, Villey IJ, Ptaszek LM, Schatz DG (2000b) Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Mol Cell 5: 97–107 [DOI] [PubMed] [Google Scholar]
  27. Grazini U, Zanardi F, Citterio E, Casola S, Goding CR, McBlane F (2010) The RING domain of RAG1 ubiquitylates histone H3: a novel activity in chromatin‐mediated regulation of V(D)J joining. Mol Cell 37: 282–293 [DOI] [PubMed] [Google Scholar]
  28. Grundy GJ, Yang W, Gellert M (2010) Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proc Natl Acad Sci USA 107: 22487–22492 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Helmink BA, Sleckman BP (2012) The response to and repair of RAG‐mediated DNA double‐strand breaks. Annu Rev Immunol 30: 175–202 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Herzog S, Reth M, Jumaa H (2009) Regulation of B‐cell proliferation and differentiation by pre‐B‐cell receptor signalling. Nat Rev Immunol 9: 195–205 [DOI] [PubMed] [Google Scholar]
  31. Horowitz JE, Bassing CH (2014) Noncore RAG1 regions promote Vbeta rearrangements and alphabeta T cell development by overcoming inherent inefficiency of Vbeta recombination signal sequences. J Immunol 192: 1609–1619 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Innes CL, Heinloth AN, Flores KG, Sieber SO, Deming PB, Bushel PR, Kaufmann WK, Paules RS (2006) ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts. Mol Cancer Res 4: 197–207 [DOI] [PubMed] [Google Scholar]
  33. Innes CL, Hesse JE, Palii SS, Helmink BA, Holub AJ, Sleckman BP, Paules RS (2013) DNA damage activates a complex transcriptional response in murine lymphocytes that includes both physiological and cancer‐predisposition programs. BMC Genomics 14: 163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Innes CL, Hesse JE, Morales AJ, Helmink BA, Schurman SH, Sleckman BP, Paules RS (2020) DNA damage responses in murine pre‐B cells with genetic deficiencies in damage response genes. Cell Cycle 19: 67–83 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ji Y, Resch W, Corbett E, Yamane A, Casellas R, Schatz DG (2010) The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141: 419–431 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Johnson K, Hashimshony T, Sawai CM, Pongubala JM, Skok JA, Aifantis I, Singh H (2008) Regulation of immunoglobulin light‐chain recombination by the transcription factor IRF‐4 and the attenuation of interleukin‐7 signaling. Immunity 28: 335–345 [DOI] [PubMed] [Google Scholar]
  37. Johnston R, White LS, Bednarski JJ (2022) Assessing DNA damage responses using B lymphocyte cultures. Methods Mol Biol 2444: 69–80 [DOI] [PubMed] [Google Scholar]
  38. Jones JM, Simkus C (2009) The roles of the RAG1 and RAG2 "non‐core" regions in V(D)J recombination and lymphocyte development. Arch Immunol Ther Exp (Warsz) 57: 105–116 [DOI] [PubMed] [Google Scholar]
  39. Kassmeier MD, Mondal K, Palmer VL, Raval P, Kumar S, Perry GA, Anderson DK, Ciborowski P, Jackson S, Xiong Y et al (2012) VprBP binds full‐length RAG1 and is required for B‐cell development and V(D)J recombination fidelity. EMBO J 31: 945–958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kim DR, Dai Y, Mundy CL, Yang W, Oettinger MA (1999) Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase. Genes Dev 13: 3070–3080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kim MS, Lapkouski M, Yang W, Gellert M (2015) Crystal structure of the V(D)J recombinase RAG1‐RAG2. Nature 518: 507–511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47: W171–W174 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lee YN, Frugoni F, Dobbs K, Walter JE, Giliani S, Gennery AR, Al‐Herz W, Haddad E, LeDeist F, Bleesing JH et al (2014) A systematic analysis of recombination activity and genotype‐phenotype correlation in human recombination‐activating gene 1 deficiency. J Allergy Clin Immunol 133: 1099–1108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lescale C, Deriano L (2017) The RAG recombinase: beyond breaking. Mech Ageing Dev 165: 3–9 [DOI] [PubMed] [Google Scholar]
  45. Loffert D, Ehlich A, Muller W, Rajewsky K (1996) Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4: 133–144 [DOI] [PubMed] [Google Scholar]
  46. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA‐guided human genome engineering via Cas9. Science 339: 823–826 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Matthews AG, Kuo AJ, Ramon‐Maiques S, Han S, Champagne KS, Ivanov D, Gallardo M, Carney D, Cheung P, Ciccone DN et al (2007) RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. Nature 450: 1106–1110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Melchers F (2015) Checkpoints that control B cell development. J Clin Invest 125: 2203–2210 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nagasawa T (2006) Microenvironmental niches in the bone marrow required for B‐cell development. Nat Rev Immunol 6: 107–116 [DOI] [PubMed] [Google Scholar]
  50. Notarangelo LD, Kim MS, Walter JE, Lee YN (2016) Human RAG mutations: Biochemistry and clinical implications. Nat Rev Immunol 16: 234–246 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Oettinger MA, Schatz DG, Gorka C, Baltimore D (1990) RAG‐1 and RAG‐2, adjacent genes that synergistically activate V(D)J recombination. Science 248: 1517–1523 [DOI] [PubMed] [Google Scholar]
  52. Purman CE, Collins PL, Porter SI, Saini A, Gupta H, Sleckman BP, Oltz EM (2019) Regional gene repression by DNA double‐Strand breaks in G1 phase cells. Mol Cell Biol 39: e00181‐19 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rajewsky K (1996) Clonal selection and learning in the antibody system. Nature 381: 751–758 [DOI] [PubMed] [Google Scholar]
  54. Rooney S, Alt FW, Lombard D, Whitlow S, Eckersdorff M, Fleming J, Fugmann S, Ferguson DO, Schatz DG, Sekiguchi J (2003) Defective DNA repair and increased genomic instability in Artemis‐deficient murine cells. J Exp Med 197: 553–565 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rooney S, Chaudhuri J, Alt FW (2004) The role of the non‐homologous end‐joining pathway in lymphocyte development. Immunol Rev 200: 115–131 [DOI] [PubMed] [Google Scholar]
  56. Sadofsky MJ, Hesse JE, McBlane JF, Gellert M (1993) Expression and V(D)J recombination activity of mutated RAG‐1 proteins. Nucleic Acids Res 21: 5644–5650 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schatz DG, Oettinger MA, Baltimore D (1989) The V(D)J recombination activating gene, RAG‐1. Cell 59: 1035–1048 [DOI] [PubMed] [Google Scholar]
  58. Silver DP, Spanopoulou E, Mulligan RC, Baltimore D (1993) Dispensable sequence motifs in the RAG‐1 and RAG‐2 genes for plasmid V(D)J recombination. Proc Natl Acad Sci USA 90: 6100–6104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Simkus C, Anand P, Bhattacharyya A, Jones JM (2007) Biochemical and folding defects in a RAG1 variant associated with Omenn syndrome. J Immunol 179: 8332–8340 [DOI] [PubMed] [Google Scholar]
  60. Simkus C, Makiya M, Jones JM (2009) Karyopherin alpha 1 is a putative substrate of the RAG1 ubiquitin ligase. Mol Immunol 46: 1319–1325 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Soodgupta D, White LS, Yang W, Johnston R, Andrews JM, Kohyama M, Murphy KM, Mosammaparast N, Payton JE, Bednarski JJ (2019) RAG‐mediated DNA breaks attenuate PU.1 activity in early B cells through activation of a SPIC‐BCLAF1 complex. Cell Rep 29: 829–843 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA‐guided endonuclease Cas9. Nature 507: 62–67 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA et al (2003) Lentivirus‐delivered stable gene silencing by RNAi in primary cells. RNA 9: 493–501 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Talukder SR, Dudley DD, Alt FW, Takahama Y, Akamatsu Y (2004) Increased frequency of aberrant V(D)J recombination products in core RAG‐expressing mice. Nucleic Acids Res 32: 4539–4549 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Tubbs AT, Dorsett Y, Chan E, Helmink B, Lee BS, Hung P, George R, Bredemeyer AL, Mittal A, Pappu RV et al (2014) KAP‐1 promotes resection of broken DNA ends not protected by gamma‐H2AX and 53BP1 in G(1)‐phase lymphocytes. Mol Cell Biol 34: 2811–2821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wang G, Dhar K, Swanson PC, Levitus M, Chang Y (2012) Real‐time monitoring of RAG‐catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex. Nucleic Acids Res 40: 6082–6096 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yurchenko V, Xue Z, Sadofsky M (2003) The RAG1 N‐terminal domain is an E3 ubiquitin ligase. Genes Dev 17: 581–585 [DOI] [PMC free article] [PubMed] [Google Scholar]

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