Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks This information is current as of September 23, 2021. Megan R. Fisher, Adrian Rivera-Reyes, Noah B. Bloch, David G. Schatz and Craig H. Bassing J Immunol 2017; 198:2943-2956; Prepublished online 17 February 2017; doi: 10.4049/jimmunol.1601639 Downloaded from http://www.jimmunol.org/content/198/7/2943

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Megan R. Fisher,*,†,‡ Adrian Rivera-Reyes,*,‡,x Noah B. Bloch,* David G. Schatz,{ and Craig H. Bassing*,†,‡,x

Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at Igk loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previ- ously reported that RAG DSBs induced at one Igk allele signal through the ataxia telangiectasia mutated (ATM) kinase to

feedback-inhibit RAG expression and RAG cleavage of the other Igk allele. In this article, we show that DSBs induced by ionizing Downloaded from radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA, but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-kB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-kB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igk loci and a chromosomally integrated substrate. Our http://www.jimmunol.org/ data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic Ag receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of monospecific lymphocytes while suppressing oncogenic Ag receptor locus translocations. The Journal of Immunology, 2017, 198: 2943–2956.

ouble-strand breaks (DSBs) are unavoidable, common, terations that cause cancer if they are not repaired or are aberrantly and hazardous genomic lesions. They are induced by repaired. Mammalian cells have evolved a conserved DNA damage endogenous factors including cellular metabolites, gene response (DDR) that coordinates DSB repair with cellular pro-

D by guest on September 23, 2021 transcription, and DNA replication, and by exogenous factors liferation and survival to maintain cellular function, preserve ge- including ionizing radiation (IR) and genotoxic drugs. DSBs can nomic integrity, and suppress malignant transformation. A central impair cellular function, induce apoptosis, or create genomic al- component of this shared DDR is the ataxia telangiectasia mutated (ATM) kinase, which is activated by DSBs (1). ATM phosphor- *Division of Cancer Pathobiology, Department of Pathology and Laboratory Medi- ylates and activates DNA repair proteins to enhance the kinetics cine, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, and fidelity of DSB repair (2). ATM also activates intracellular † Philadelphia, PA 19104; Immunology Graduate Group, Perelman School of Medi- signaling pathways that maintain cellular survival and halt DNA cine at the University of Pennsylvania, Philadelphia, PA 19104; ‡Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, synthesis as cells attempt to repair DSBs, and promote apoptosis if Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA x DSBs are not repaired (1). For example, ATM phosphorylates the 19104; Cancer Biology Program of the Cell and Molecular Biology Graduate Group, NF-kB essential modulator (Nemo) protein to activate NF-kB– Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104; and {Department of Immunobiology, Yale University School of Medicine, mediated transcription of antiapoptotic genes (3). In parallel, Howard Hughes Medical Institute, New Haven, CT 06520 ATM-dependent phosphorylation of the Tp53 protein activates Received for publication September 22, 2016. Accepted for publication January 16, transcription of cell cycle checkpoint and proapoptotic genes (1). 2017. The importance of the conserved mammalian DDR is highlighted This work was supported by the Training Program in Rheumatic Diseases of the by the increased predisposition of humans and mice lacking ATM University of Pennsylvania (Grant T32 AR007442 to M.R.F.), the National Research Service Award (Grant F31 CA183551 to M.R.F.), the Training Program in Cell and protein to oncogenic genomic instability (4–8). Molecular Biology of the University of Pennsylvania (Grant GM007229 T32 to Despite their inherent danger, DSBs are essential for mammalian A.R.-R), the National Institutes of Health (Grant R37 AI32524 to D.G.S.; R01 Grant biology. The programmed induction of DSBs by tissue-specific AI112621 to C.H.B.), and the Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia (to C.H.B.). proteins is necessary to establish the genetic diversity that drives Address correspondence and reprint requests to Dr. Craig H. Bassing, Children’s evolution and provides adaptive immunity (9, 10). A paradigm for Hospital of Philadelphia, 4054 Colket Translational Research Building, 3501 Civic this concept is the assembly of Ag receptor (AgR) gene V region Center Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] exons via recombination of variable (V), diversity (D), and The online version of this article contains supplemental material. joining (J) gene segments in developing B and T cells (11). Abbreviations used in this article: AgR, Ag receptor; ATM, ataxia telangiectasia The lymphocyte-specific RAG endonuclease catalyzes V(D)J re- mutated; BM, bone marrow; D, diversity; DDR, DNA damage response; DSB, double-strand break; EU, ethynyl uridine; ERag, B cell–specific transcriptional en- combination in G1 phase cells by cleaving at recombination signal hancer of the Rag1/Rag2 locus; IR, ionizing radiation; J, joining; NEB, New England sequences (RSSs) that flank all V, D, and J segments (12). The Biolabs; NHEJ, nonhomologous end-joining; RT-PCR, real-time PCR; RSS, recom- resolution of RAG DSBs by nonhomologous end-joining (NHEJ) bination signal sequence; V, variable. factors generates V(D)J coding joins and RSS signal joins (2). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 These V(D)J exons and downstream C region exons comprise www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601639 2944 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION assembled AgR genes. The large number of possible V(D)J joining cleavage and demonstrated that this regulation involves ATM- events and the inherent imprecision of coding join formation coop- dependent DDR signaling pathways that repress RAG expres- erate to establish AgR gene diversity. The essential role for RAG sion and Igk recombination potential. DSBs in establishing immunity is emphasized by mutations of RAG1, Because DSBs induced in pre-B cells by IR or etoposide can activate RAG2, RSSs, or NHEJ factors that impair lymphocyte development, expression of some lymphocyte-specific genes induced by RAG DSBs restrict AgR gene repertoires, and cause fatal SCID (13–15). (22, 28), we hypothesized that any type of DSB can signal inhibition RAG DSBs and the DDR cooperate to promote and police AgR of RAG expression and V(D)J recombination. In this study, we gene assembly and differentiation of lymphocytes in the bone demonstrate that DSBs induced in primary lymphocytes by IR or marrow (BM; B lymphocytes) and thymus (T lymphocytes). The etoposide signal through ATM and Nemo to transcriptionally repress assembly of IgH or TCRb genes in pro-B or pro-T cells, re- Rag1 expression and inhibit RAG cleavage of Igk loci and RSSs spectively, generates pre-BCR or pre-TCR complexes that signal integrated at another genomic location. While we were preparing our cellular proliferation and differentiation into pre-B or pre-T cells manuscript, the Guikema laboratory (29) reported that DSBs induced (16). Similarly, assembly of IgL (Igk or Igl) or TCRa genes in in immortalized and transformed human and mouse pre-B cell lines pre-B or pre-T cells, respectively, forms BCR and ab TCR AgRs signal via ATM and the FOXO1 transcription factor to inhibit RAG that signal cellular differentiation or apoptosis (16). Autoreactive expression and activity. We discuss how our study corroborates the AgRs induce receptor editing through further IgL recombination central findings of the Guikema laboratory and present novel data that or apoptosis, whereas innocuous AgRs halt Rag1 and Rag2 tran- provide further insights into the mechanisms and the scope by scription and drive differentiation of conventional B and ab which lymphocytes direct DSB-induced feedback inhibition of V(D)J

T cells (17). In pro-T cells, the assembly of TCRg and TCRd recombination. We also outline the implications of both studies to Downloaded from genes also occurs and, when successful, produces gd TCRs that mechanisms that orchestrate the differentiation of monospecific terminate Rag1 and Rag2 transcription and signal differentiation lymphocytes while suppressing oncogenic AgR locus translocations. of gd T cells (18). RAG cleavage of AgR loci activates the ATM kinase (19), which phosphorylates DSB repair proteins to prevent Materials and Methods degradation and enhance joining of RAG-liberated DNA ends (2). Mice

ATM also activates Tp53 to trigger the G1/S cell cycle checkpoint, All mice used within this study were housed, bred, and used under http://www.jimmunol.org/ inhibiting cells with RAG DSBs from entering S phase, where pathogen-free conditions at the Children’s Hospital of Philadelphia. All DNA replication-associated DSBs increase the probability for experiments were performed using 4- to 6-wk-old mice using both male AgR locus translocations (20, 21) At least in pre-B cells, RAG and female mice. Experimental mice were euthanized by exposure to CO2 followed by cervical dislocation. Animal husbandry and experiments were DSBs signal through both ATM-dependent and ATM-independent performed in accordance with national guidelines and regulations and were pathways to activate a broad multifunctional genetic program that approved by the Children’s Hospital of Philadelphia Institutional Animal includes genes known to regulate lymphocyte differentiation and Care and Use Committee. The EmBCL2 (30), Rag1D708A (31), Mb1Cre+ flox/flox + flox/flox + flox/flox lymphocyte-specific processes (22). One of these pathways involves Atm (24), VavCre p53 (32), and Mb1Cre Nemo (33) mice were on a mixed 129S1/SvImJ and C57BL/6 background, whereas ATM-dependent Nemo phosphorylation, which induces NF-kB– 2 2 2 2 Rag1 / (34) and Rag2 / (35) mice were on the C57BL/6 background. by guest on September 23, 2021 mediated transcription of the antiapoptotic Pim2 gene to prolong The wild-type control mice were of the appropriate background. the survival of pre-B cells recombining IgL genes (22, 23). Recent work from our laboratory and the Sleckman laboratory Irradiation (24) has revealed that lymphocyte-specific RAG DSBs cooperate All mice were subject to irradiation using an XRAD320 X-ray irradiator with common DDR signaling pathways to transiently feedback- (Precision X-Ray) and a dosage rate of 0.74 Gy/min. Primary cells and cell inhibit AgR gene assembly. We demonstrated that RAG cleavage lines were subject to irradiation using a Gammacell 1000 Cs-137 irradiator (Nordion) and a dosage rate of 1.8 Gy/min. of one Igk allele in primary pre-B cells signals via the ATM kinase to suppress RAG cleavage of the other allele (24). ATM also Real-time PCR quantification of Rag1, Rag2, and p21 mRNAs downregulates the levels of Rag1 and Rag2 mRNA and Rag1 Nonirradiated and irradiated cells were harvested at indicated time points protein, as well as mRNA and protein levels of Gadd45a (24), and immediately lysed in TRIzol (Life Technologies) and processed which had been shown to signal Rag1 and Rag2 transcription in a according to the manufacturer’s instructions. Isolated RNA was treated pre-B cell line (25). However, the asynchronous induction of RAG with DNase (Promega) according to manufacturer’s directions, primed DSBs at Igk alleles precluded investigating the kinetics or with random nonamers (New England Biolabs [NEB]), and reverse tran- scribed with M-MuLV (NEB) to generate cDNA. The cDNAs were then mechanism of RAG downregulation or a potential mechanistic used as a template for real-time PCRs (RT-PCRs) performed with SYBR link between suppression of Gadd45a protein and repression of green mastermix (Applied Biosystems) and run on an Applied Biosystems Rag1 and Rag2 mRNA. We proposed that RAG DSB-induced 7500 Fast machine. Values were normalized to housekeeping genes as in- feedback inhibition of RAG expression limits simultaneous dicated, and fold change was determined by the DD cycle threshold method. The primers used for RT-PCRs are listed in Supplemental Table I. When RAG cleavage of distinct AgR alleles, helping to enforce mono- pre-B cells were subject to irradiation, nonirradiated pre-B cells also were allelic assembly and expression (allelic exclusion) of Igk, IgH, transported to the irradiator, but not exposed to IR along with irradiated and TCRb genes, and to suppress oncogenic IgH, TCRb, and samples. The irradiated and nonirradiated pre-B cells were each placed back TCRd translocations during proliferation of pro-B and pro-T cells into culture until they were harvested. Because pre-B cells were transported (24, 26). Consistent with this idea, we showed that mice lacking to and from the irradiator at room temperature, the basal levels of Rag1, Rag2, and p21 mRNAs were lower at 1 h after the irradiation time point than ATM develop higher than normal frequencies of lymphocytes with at 4 h after the irradiation time point. To inactivate the ATM kinase, we biallelic expression and/or translocations of Igk, IgH, or TCRb added the KU55933 ATM kinase inhibitor to media at a concentration loci (26). The Sleckman laboratory (27) found that RAG DSBs of 15 mM. To block new protein synthesis, we added the cycloheximide induced at one Igk allele signal via ATM to activate expression of ribosome inhibitor to media at a concentration of 10 mg/ml. the hematopoietic-specific SpiC transcriptional repressor. SpiC Primary pre-B cell cultures inhibits RAG cleavage of the other Igk allele by suppressing Primary BM cells were harvested by flushing BM from all leg bones of at least transcription-linked accessibility of Jk gene segments to the RAG four mice of the appropriate genotype for each experiment. These BM cells endonuclease (27). This body of work established the concept of were cultured for 3–5 d in RPMI 1640 supplemented with 10% FBS, 10 mM DSB-induced feedback inhibition of Igk recombination by RAG HEPES, 13 nonessential amino acids, 1 mM L-glutamine, 1 mM sodium The Journal of Immunology 2945 pyruvate, 100 U/ml penicillin-streptomycin, 30 mM 2-ME, and 50 ng/ml IL-7. with PE-conjugated anti-human CD4 Ab (clone RPA-T4; BD Pharmingen) Cells were plated at a density of 5 million cells per milliliter of media. Each to stain cells containing the substrate. Flow cytometry was conducted using day, cells were harvested and put back into culture in fresh media at a density an LSR-II flow cytometer (BD Biosciences), and data were analyzed using of 5 million cells per milliliter. To induce G1 arrest and activate transcription FlowJo 10. of Rag1 and Rag2 by IL-7 withdrawal, we pelleted cells by centrifugation, resuspended them in the same media lacking IL-7 at a density of 5 million Statistical analyses cells per milliliter, and treated them as described for each experiment. All statistics were performed using GraphPad Prism 5 or Prism 7 software. Western blot analysis of Rag1, Rag2, and actin proteins Pre-B cells were washed with PBS and resuspended in ice-cold lysis buffer Results (20 mM Tris [pH 7.4], 20 mM glycerol phosphate, 10 mM sodium The suppression of Rag1 and Rag2 mRNA in response to IR is a orthovanadate, 10% glycerol, 0.5 mM EDTA, 0.5 mM MgCl2, 200 mM prevalent response of immature lymphocytes NaCl, and 0.2% Triton-X) and then sonicated at intervals of 30 s on and 30 s off for 8 min at 4˚C. The sonicated cells were incubated on ice for We first sought to determine the ability of DSBs induced by IR 10 min and then centrifuged to remove insoluble material. Laemmli buffer to suppress Rag1 and Rag2 expression in developing B and was added; then samples were boiled for 5 min. Lysates from equivalent numbers of pre-B cells were loaded and run on NuPAGE tris-glycine gels T lymphocytes. Because we discovered that the DDR of immature (Life Technologies). Electrophoresed proteins were transferred to lymphocytes frequently is abnormal in immortalized cell lines and in Immobilon-FL PVDF membrane (EMD Millipore). Membranes were primary cells cultured ex vivo (41), we conducted initial experiments blocked with Odyssey blocking buffer (LI-COR) according to manufac- by exposing live mice to IR. To limit apoptosis of immature turer’s instructions. Abs used are anti-Rag1 or anti-Rag2 mAb (36), anti- lymphocytes, we used EmBCL2 mice with transgenic expression of actin Ab (AV40173; Sigma), and anti-Gadd45a (sc-797; Santa Cruz). Blots were washed and incubated with appropriate IRDye800 secondary Abs the antiapoptotic BCL2 protein in all B and T lineage cells. We Downloaded from (LI-COR) according to the manufacturer’s instructions. After washing, exposed these mice to IR and harvested cells from their thymuses and blots were scanned on an Odyssey infrared scanner (LI-COR). BM 1 or 4 h later. We conducted RT-PCR on RNA isolated from Click-It analysis of Rag1 and Rag2 mRNA turnover and Rag1 these cells or corresponding cells of nonirradiated EmBCL2 mice. We and Rag2 transcription quantified levels of Rag1 and Rag2 mRNA relative to levels of the T lineage–specific Lck mRNA for thymus or the B lineage–specific These assays were conducted using Click-It Nascent RNA Capture kits (Life Cd19 mRNA for BM. To control for the induction of DSBs, we http://www.jimmunol.org/ Technologies). For mRNA turnover assays, ethynyl uridine (EU) was added to medium of IL-7 withdrawn pre-B cell cultures at a final concentration of quantified mRNA levels of the p21 cyclin–dependent inhibitor gene 0.2 mM for the final 16 h of culture time. Pre-B cells were washed, placed whose transcription is activated by ATM and Tp53 after DSBs (1). into media lacking EU, and then split into pools that were immediately We detected lower levels of Rag1 and Rag2 mRNAs and higher irradiated or left nonirradiated. Cell pools were collected for RNA isolation levels of p21 mRNA in BM from irradiated EmBCL2 mice as immediately before EU removal or at indicated times after EU removal or m EU removal and irradiation. For transcriptional assays, pre-B cells were compared with BM from nonirradiated E BCL2 mice(Fig.1A).The grown in media lacking EU. Cultures were split into pools that were either levels of Rag1 mRNA were reduced by .50%at1and4hafter irradiated or left nonirradiated. Immediately after irradiation of some pools, EU exposure to 10 Gy of IR, whereas levels of Rag2 mRNA were re- was added to the media of all pools at a final concentration of 0.5 mM. After the duced by ∼50% at both times (Fig. 1A), indicating that immature indicated times, cells were collected for RNA isolation. For both assays, RNA B lymphocytes rapidly inhibit Rag1 and Rag2 expression in response by guest on September 23, 2021 was isolated using TRIzol (Life Technologies) according to the manufacturer’s instructions. Click chemistry and streptavidin pulldown of EU-labeled RNA to IR. In contrast, we observed no decrease in the level of Rag1 or were performed according to the Click-It Nascent RNA Capture kit’s in- Rag2 mRNA in thymuses from irradiated EmBCL2 mice relative to structions. Pulled-down RNA was reverse transcribed and analyzed by RT- thymuses from nonirradiated EmBCL2 mice, despite induction of PCR as described earlier. The loading controls for mRNA turnover and DSBs as reflected by elevated p21 mRNA levels (Fig. 1B). Indeed, transcription experiments were 18S RNA and Hprt mRNA, respectively. the levels of Rag1 and Rag2 mRNA each were increased by ∼40% at Abelson pro-B cell cultures 4 h after exposure to 10 Gy of IR (Fig. 1B). We corroborated that IR The EmBCL2 (A2) (37) and Artemis2/2EmBCL2 (Art2.1) (38) Abelson rapidly downregulates Rag1 and Rag2 mRNA in BM, but not thy- transformed and immortalized pro-B cell lines were previously described. muses, of wild-type mice that lack the prosurvival EmBCL2 trans- We made the EmBCL2-pINV (39) Abelson pro-B cell line using the same gene (Fig. 1C, 1D). Collectively, these data suggest that DSBs procedures that we previously used to generate cell lines with recombi- induced by IR inhibit Rag1 and Rag2 expression in developing nation substrates (40). Abelsons pro-B cells were cultured in RPMI media supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, and B lymphocytes, but not developing T lymphocytes. However, con- 30 mM 2-ME. To induce G1 cell cycle arrest, differentiation into pre- sidering that BM and thymuses each contain a much greater number B cells, expression of Rag1 and Rag2 mRNA, and V(D)J recombination, of prelymphocytes than prolymphocytes, this experimental approach we added 5 mM STI571 to the culture media. To induce DSBs by geno- likely is not sensitive enough to determine whether prolymphocytes toxic drugs, we added etoposide to the media at a concentration of 10 mg/ml, downregulate Rag1 and Rag2 expression in response to DSBs. or bleomycin was added at a concentration of 5 mM. We next sought to determine the effects of DSBs on Rag1 and Southern blot analysis Rag2 expression in primary pro-B and pro-T cells. For this 2/2 2/2 Southern blotting was performed as described previously (38). In brief, 15– purpose, we turned to Rag1 and Rag2 mice in which 20 mg of genomic DNA was digested with Sac1-HF (NEB) and EcoR1-HF lymphocyte development is blocked at the pro-B and pro-T cell (NEB). Southern blot membranes were probed with a 39Jk probe and then a stages (34, 35). The Rag12/2 mice have a drug-resistance gene 59Hprt probe as a loading control. The intensities of bands were quantified 2/2 driven by a constitutive promoter in place of Rag1 coding se- using ImageJ software (National Institutes of Health). For Artemis 2/2 EmBLC2 cells, the percentage of Jk cleavage at each time point was cal- quences, whereas the Rag2 mice have a loxP site in place of culated by dividing the total intensities of Jk coding end bands by the Rag2 coding sequences. We observed decreased levels of Rag1 combined intensities of the germline Jk band and Jk coding end bands (40). mRNA in BM from irradiated Rag22/2 mice relative to BM from For EmBLC2 cells, the percentage of Jk cleavage at each time point was nonirradiated Rag22/2 mice (Fig. 1E). The levels of Rag1 mRNA k calculated by dividing the intensity of the germline J band by the intensity 2/2 ∼ of the 59Hprt control band. in Rag2 BM were reduced 75% at 1 h after 10 Gy of IR and ∼90% at 4 h after 10 Gy of IR. Notably, we detected lower levels of Flow cytometry Rag1 mRNA in thymuses from irradiated Rag22/2 mice as com- 2/2 For analysis of GFP expression in EmBCL2-pINV cells, cells were washed pared with thymuses from nonirradiated Rag2 mice (Fig. 1F). 2 2 in FACS buffer (PBS with 3% FCS and 0.25 mM EDTA) and then stained The levels of Rag1 mRNA in Rag2 / thymuses were reduced 2946 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION Downloaded from http://www.jimmunol.org/

FIGURE 1. Immature B and T lymphocytes suppress Rag1 and Rag2 expression in response to IR. (A) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in BM from nonirradiated EmBCL2 mice or EmBCL2 mice at indicated times after exposure to 10 Gy of IR. Data are from one experiment with three to four mice for each time point. (B) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in thymuses from nonirradiated EmBCL2 mice or EmBCL2 mice at indicated times after exposure to 10 Gy of IR. Data are from three independent experiments including a total of three to six mice per time point. (C) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in BM from nonirradiated wild-type mice at indicated times after exposure to 10 Gy of IR.

Data are from two independent experiments including a total of four to five mice per time point. (D) RT-PCR quantification of Rag1, Rag2, and p21 mRNA by guest on September 23, 2021 in thymus from nonirradiated wild-type mice or wild-type mice at indicated times after exposure to 10 Gy of IR. Data are from two independent ex- periments including a total of four to five mice per time point. (E) RT-PCR quantification of Rag1 and p21 mRNA in BM from nonirradiated Rag22/2 mice or Rag22/2 mice at indicated times after exposure to 10 Gy of IR. Data are from one experiment with three or four mice per time point. (F) RT-PCR quantification of Rag1 and p21 mRNA in thymus from nonirradiated Rag22/2 mice or Rag22/2 mice at indicated times after exposure to 10 Gy of IR. Data are from one experiment with three or four mice per time point. (A–F) Data averages are shown with error bars indicating SEM. The p values were calculated using Dunnett’s posttest after ANOVA: *p , 0.05, **p , 0.01, ***p , 0.001.

∼40% at 1 and 4 h after 10 Gy IR. Consistent with these findings, then isolated RNA at 1 or 4 h after irradiation. In parallel, we we observed that IR downregulated Rag2 mRNA in BM and thymus of harvested RNA from nonirradiated pre-B cells. We used RT-PCR Rag12/2 mice to a similar extent as Rag1 mRNA in Rag22/2 mice to quantify Rag1 and Rag2 mRNA levels relative to actin mRNA (Supplemental Fig. 1). The results of our experiments with Rag12/2 levels. We also quantified p21 mRNA to confirm the induction of and Rag22/2 mice suggest that DSBs downregulate Rag1 and Rag2 DSBs. We detected lower levels of Rag1 and Rag2 mRNA and expression in primary pro-B and pro-T cells. higher levels of p21 mRNA in irradiated EmBCL2 pre-B cells as compared with nonirradiated EmBCL2 pre-B cells (Fig. 2A). The The induction of DSBs in pre-B cells downregulates levels of levels of Rag1 and Rag2 mRNA each were reduced ∼80% at 1 and Rag1 and Rag2 mRNAs 4 h after exposure to IR (Fig. 2A), indicating that pre-B cells To validate that DSBs induced by IR in primary pre-B cells rapidly inhibit Rag1 and Rag2 expression in response to IR. downregulate Rag1 and Rag2 expression, we turned to the ex Although our data suggest that DSBs induced by IR inhibit Rag1 vivo culture system wherein we discovered that RAG DSBs in- and Rag2 expression, they cannot rule out that other types of hibit Rag1 and Rag2 expression (24). The culture of primary cellular damage caused by IR promote this response. To address mouse BM in media containing IL-7 cytokine selectively pro- this issue, instead of exposing pre-B cells to IR, we exposed cells motes survival and proliferation of pre-B cells, such that within a to drugs that predominantly induce DSBs. Bleomycin is a geno- few days .95% of cells in culture are pre-B cells (42). IL-7 also toxic drug that induces DSBs throughout the cell cycle, but also suppresses Rag1 and Rag2 expression (43). After removal of IL-7, damages RNA (44). We observed lower levels of Rag1 and Rag2 pre-B cells arrest in G1 phase and activate Rag1 and Rag2 tran- mRNAs and higher levels of p21 mRNA in EmBCL2 pre-B cells scription (42). Expression of the EmBCL2 transgene enables pre- treated with bleomycin relative to EmBCL2 pre-B cells treated B cells to survive and express Rag1 and Rag2 for several days with only vehicle (Fig. 2B). The levels of Rag1 and Rag2 mRNA after IL-7 removal (42). We cultured pre-B cells from EmBCL2 each were reduced ∼60–80% at 1 and 4 h with treatment with mice, removed IL-7 for 48 h to arrest cells in G1 phase and induce bleomycin (Fig. 2B), indicating that DSBs induced in pre-B cells Rag1 and Rag2 transcription, exposed cells to 4 Gy of IR, and rapidly inhibit Rag1 and Rag2 expression. Etoposide is a genotoxic The Journal of Immunology 2947 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. DSBs induced in pre-B cells downregulate Rag1 and Rag2 mRNA and Rag1 protein. (A) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in nonirradiated EmBCL2 pre-B cells or irradiated EmBCL2 pre-B cells at indicated times after exposure to 4 Gy of IR. Data are from 11 inde- pendent experiments. (B) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in EmBCL2 pre-B cells or EmBCL2 pre-B cells at indicated times after addition of 5 mM bleomycin to culture media. Data are from three independent experiments. (C) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in EmBCL2 pre-B cells or EmBCL2 pre-B cells at indicated times after addition of 10 mg/ml etoposide to culture media. Data are from three independent experiments. (D) Representative Western blot analysis and graphs depicting quantification of Rag1 and Rag2 protein in nonirradiated EmBCL2 pre-B cells or irradiated EmBCL2 pre-B cells at indicated times after exposure to 4 Gy of IR. Data are from eight independent experiments. (E) Representative Western blot analysis and graphs depicting quantification of Rag2 and c-Myc protein in cycloheximide-treated EmBCL2 pre-B cells at indicated times after cy- cloheximide addition. Data are from three independent experiments. (A–E) Data are normalized to 1.0 for nonirradiated cells within each experiment. For irradiated cells, data averages are shown with error bars indicating SEM. The p values calculated using Dunnett’s posttest after ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. drug whose only demonstrated biological effect is that it inhibits of DSB activates DSB-induced feedback inhibition of Rag1 and religation of DNA by type II topoisomerase enzymes, causing ac- Rag2 expression. cumulation of DSBs (45). Although etoposide causes DSBs mainly DSBs induced in pre-B cells inhibit expression of Rag1 protein, in S phase cells (45), this genotoxic agent does induce DSBs in G1 arrested pre-B cells (46). We detected lower levels of Rag1 and but not Rag2 protein Rag2 mRNAs and higher levels of p21 mRNA in EmBCL2 pre- For DSB-induced feedback inhibition of Rag1 and Rag2 expres- B cells treated with etoposide relative to EmBCL2 pre-B cells sion to suppress V(D)J recombination, Rag1 and/or Rag2 protein treated with only vehicle (Fig. 2C). The levels of Rag1 and Rag2 levels must be diminished as a result of decreased Rag1 and Rag2 mRNA each were reduced ∼80%at1and4hwithtreatmentwith mRNA. Previous studies reported that the Rag1 and Rag2 proteins etoposide (Fig. 2C), indicating that DSBs induced in pre-B cells each have a half-life of ∼10–12 min (29, 47, 48). Considering rapidly inhibit Rag1 and Rag2 expression. Considering that IR, these reports and our observed kinetics and extents of Rag1 and bleomycin, etoposide, and RAG cleavage of Igk loci (24) each Rag2 mRNA loss in response to IR, one would predict appreciable suppress Rag1 and Rag2 mRNA levels, we conclude that any type decreased levels of Rag1 and Rag2 protein at 1 h after irradiation. 2948 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION

To confirm this prediction, we conducted Western blot analysis of IL- each have a half-life of ∼40 min (Fig. 3A), similar to published 7–withdrawn primary pre-B cells at 1 and 4 h after their exposure to results using nuclear run-on assays on primary pre-B cells (53). At 4 Gy of IR. In parallel, we assayed Rag1 and Rag2 protein in non- all times assayed, we detected no difference in the levels of labeled irradiated pre-B cells. We also conducted Western blot analysis for Rag1 mRNA between irradiated and nonirradiated cells (Fig. 3A), actin protein and quantified Rag1 and Rag2 protein levels relative to indicating that pre-B cells do not increase turnover of Rag1 mRNA actin protein. We observed reduced levels of Rag1 protein in irra- in response to DSBs. In contrast, we observed lower levels of la- diated EmBCL2 pre-B cells as compared with nonirradiated EmBCL2 beled Rag2 mRNA at 30 and 45 min after irradiation (Fig. 3A), pre-B cells (Fig. 2D). Consistent with our prediction, Rag1 protein reflecting that DSBs induced in pre-B cells decrease the half-life of was reduced ∼50%at1hafter4GyofIRand∼75%at4hafter4 Rag2 mRNA from ∼40 to ∼30 min. To investigate Rag1 and Rag2 Gy of IR. In marked contrast, we detected no change in Rag2 protein transcription, we added EU to IL-7–withdrawn EmBCL2 pre-B cells levels after irradiation of EmBCL2 pre-B cells (Fig. 2D), which is not immediately after exposure to 4 Gy of IR. We also added EU to consistent with our prediction. Regardless, because both Rag1 and nonirradiated IL-7–withdrawn EmBCL2 pre-B cells. Over time, we Rag2 are required for RAG endonuclease activity (49), the down- measured the amounts of newly synthesized, EU-labeled Rag1 and regulation of Rag1 protein levels in response to the induction of Rag2 mRNA and the loading control Hprt mRNAs. In this exper- DSBs could be sufficient to inhibit V(D)J recombination. iment, the balance of transcription and mRNA turnover determines the amount of a labeled mRNA. By 45 min after irradiation, we Endogenous Rag2 protein in primary pre-B cells has an observed near-complete loss of labeled Rag1 and Rag2 mRNAs in immeasurably long half-life irradiated cells relative to nonirradiated cells (Fig. 3B). Considering

We found the retention of Rag2 protein after the loss of Rag2 the ∼30- to 40-min turnover of Rag1 and Rag2 mRNAs in irradi- Downloaded from mRNA interesting, particularly because it was not predicted from ated pre-B cells (Fig. 3A), the kinetics and extents by which EU- the published half-life of Rag2 protein. The possible explanations labeled Rag1 and Rag2 mRNAs are lost in irradiated cells suggests of our finding are that the basal half-life of Rag2 protein in primary that DSBs cause an immediate halt of Rag1 and Rag2 transcription. pre-B cells is much longer than ∼45 min and/or DSBs induced in To investigate whether DDR activation in immature lymphocytes primary pre-B cells increase the half-life of Rag2 protein. The leads to repression of genes nonessential to the genotoxic stress

Rag2 protein is expressed in G1 but not in subsequent cell cycle response, we also measured the amount of newly synthesized, EU- http://www.jimmunol.org/ phases because of its phosphorylation and consequent degradation labeled Wasp mRNA, because Wasp has no documented role in upon S phase entry (48, 50, 51), supporting the possibility that the DDR and irradiation of primary pre-B cells has no effect on Rag2 protein might be very stable in G1 phase cells. The published steady-state mRNA levels (data not shown). Our analysis reveals half-life of Rag2 protein is only 10–12 min; however, this value that irradiation of primary pre-B cells has minimal effect on the was obtained by measuring asynchronously cycling nonlymphoid synthesis of Wasp mRNA (Fig. 3B). Thus, we conclude that Rag1 cell lines with ectopic expression of Rag2 (48, 51). Therefore, we and Rag2 transcription are specifically targeted by the DDR, and investigated the basal half-life of Rag2 protein in G1-arrested pri- not simply part of a generalized inhibition of transcription in the mary pre-B cells. We conducted Western blot analysis of IL-7– presence of widespread DNA damage. withdrawn primary pre-B cells at increasing times after addition of Consistent with the rapid timeframe of Rag1and Rag2 transcrip- by guest on September 23, 2021 cycloheximide to block new protein synthesis. As a positive control tional suppression, inhibition of new protein synthesis by cyclohex- for inhibition of protein synthesis, we also conducted Western blot imide treatment does not prevent the loss of Rag1 and Rag2 mRNA analysis of the c-Myc protein, which has a half-life of ,1h(52). levels after irradiation of EmBCL2 pre-B cells (Fig. 3C). This finding Strikingly, we detected no loss of Rag2 protein at any time point suggests that DSBs suppress Rag1 and Rag2 transcription through after cycloheximide addition, despite rapid loss of c-Myc protein factors constitutively expressed in primary pre-B cells. Collectively, (Fig. 2E). In this context, we could not calculate the half-life of these data are consistent with repression of Rag1 and Rag2 tran- Rag2 protein because we detected no loss of Rag2 protein levels at scription being the predominant mechanism by which pre-B cells 24 h after cycloheximide (Fig. 2E), after which pre-B cells begin to signal DSB-induced loss of Rag1 and Rag2 expression. die of lack of new protein synthesis. Because we could not detect loss of Rag2 protein in nonirradiated cells, we were not able to DSB-induced downregulation of Rag1 and Rag2 expression ascertain the potential effect of DSBs on Rag2 protein stability. requires the ATM kinase We next launched a line of research to elucidate the factors by which DSBs induced in pre-B cells signal rapid transcriptional DSBs induced in primary pre-B cells suppress Rag1 and Rag2 repression of Rag1 and Rag2 transcription. We previously reported that RAG DSBs induced in We next sought to elucidate mechanisms by which DSBs cause pre-B cells signal via ATM to downregulate Rag1 and Rag2 ex- downregulation of Rag1 and Rag2 mRNA. This loss of mRNA pression (24). Therefore, we investigated the role of ATM through could be caused by increased turnover of Rag1 and Rag2 mRNAs chemical inhibition of the ATM kinase or genetic inactivation of the and/or repression of Rag1 and Rag2 transcription. We used Click- ATM protein. First, we added the ATM kinase inhibitor KU55933 or iT mRNA capture technology to investigate these possibilities. vehicle only at the same time that we removed IL-7 from EmBCL2 Click-iT mRNA capture incorporates ethylene uridine (EU) into pre-B cell cultures; 48 h later, we exposed cells to 4 Gy of IR or left RNAs during transcription; EU-labeled RNAs are conjugated with them untreated and then harvested cells for RT-PCR quantification biotin, isolated by streptavidin beads, and quantified by RT-PCR. of Rag1 and Rag2 mRNA. We also quantified p21 mRNA as a To measure turnover of Rag1 and Rag2 mRNAs in irradiated and control for both induction of DSBs and inactivation of the ATM nonirradiated primary pre-B cells, we added EU to the media of kinase, because genetic deletion of ATM impairs but does not block EmBCL2 pre-B cell cultures for the last 16 h of IL-7 withdrawal to the ability of DSBs to induce p21 mRNA levels (54). We detected fully label existing mRNAs. We washed out EU immediately ∼2- to 3-fold higher levels of Rag1 and Rag2 mRNAs in nonirra- before exposure of cells to 4 Gy of IR to block further EU in- diated cells treated with KU55933 versus vehicle only (Fig. 4A), corporation so that we could measure the amounts of EU-labeled likely reflecting ATM-dependent suppression of Rag1 and Rag2 Rag1 and Rag2 mRNA remaining over time in irradiated and transcription from DSBs induced by RAG during Igk recombination nonirradiated cells. We found that the Rag1 and Rag2 mRNAs and by other transcription and cellular metabolites. Notably, the The Journal of Immunology 2949 Downloaded from http://www.jimmunol.org/

FIGURE 3. DSBs induced in primary pre-B cells immediately repress Rag1 and Rag2 transcription. (A) RT-PCR quantification of EU-labeled Rag1 and m m

Rag2 mRNA levels relative to EU-labeled 18S RNA levels in nonirradiated E BCL2 pre-B cells or irradiated E BCL2 pre-B cells at indicated times after by guest on September 23, 2021 EU washout and/or exposure to 4 Gy of IR. Data are from four independent experiments. Data averages are shown with error bars indicating the SEM. Prism 5 was used to calculate best-fit curves, half-lives, and p values. (B) RT-PCR quantification of EU-labeled Rag1, Rag2, and Wasp mRNA levels relative to EU-labeled Hprt mRNA levels in nonirradiated EmBCL2 pre-B cells and irradiated EmBCL2 pre-B cells at indicated times after addition of EU and exposure to 4 Gy of IR. Data are presented as the ratio of relative levels of each mRNA in irradiated cells compared with nonirradiated cells. The gray line represents a value of 1, which would indicate that IR had no effect on the transcription rate of a gene. Data are from four independent experiments. The p values for whether the average ratio at each time point is different from 1.0 were determined using one-tailed t test with Bonferroni’s correction for multiple testing. (C) RT-PCR quantification of Rag1 and Rag2 mRNA in cycloheximide-treated nonirradiated EmBCL2 pre-B cells or irradiated EmBCL2 pre-B cells at indicated times after exposure to 4 Gy of IR. Data are from three independent experiments. Data averages are shown with error bars indicating the SEM. The p values were calculated by t test with Bonferroni’s correction for multiple testing. *p , 0.05, ***p , 0.001. addition of KU55933 also blocked downregulation of Rag1 and tein (24). Because Gadd45a had been shown to signal Foxo1- Rag2 mRNA after irradiation (Fig. 4A). Next, we analyzed primary dependent transcription of Rag1 and Rag2 in an immortalized pre-B cells cultured from Mb1Cre+Atmflox/flox mice with B lineage– mouse pre-B cell line (25), our findings supported a model specific conditional deletion of Atm initiating at the pro-B cell stage wherein DSBs suppress Rag1 and Rag2 transcription through (24). Unirradiated Mb1Cre+Atmflox/flox IL-7–withdrawn pre-B cells ATM-mediated downregulation of Gadd45a mRNA. To investi- expressed ∼2-fold higher levels of Rag1 and Rag2 mRNAs com- gate this model, we studied the effects of DSBs induced by IR in pared with EmBCL2 pre-B cells (data not shown), possibly reflecting primary pre-B cells on Gadd45a mRNA levels. Although we ATM-dependent inhibition of Rag1 and Rag2 expression from found that Gadd45a mRNA levels are decreased after irradiation DSBs. Moreover, irradiation of Mb1Cre+Atmflox/flox pre-B cells of EmBCL2 pre-B cells (Supplemental Fig. 2A, 2B), the slow caused no change in Rag1 and Rag mRNA levels relative to non- kinetics and limited extent of this response suggest that down- irradiated Mb1Cre+Atmflox/flox pre-B cells (Fig. 4B). The retention of regulation of Gadd45a mRNA is not a driving force in sup- Rag1 and Rag2 mRNA from 1 to 4 h after irradiation of pre-B cells pressing Rag1 and Rag2 expression (compare Fig. 3B and that lack ATM indicates that IR-induced DSBs signal through ATM Supplemental Fig. 2B). Consistent with this notion, we observed to downregulate Rag1 and Rag2 expression. that ectopic retroviral expression of Gadd45a in a mouse pre- B cell line does not prevent DSB-induced inhibition of Rag1 and DSB-induced Rag1 and Rag2 suppression does not depend on Rag2 expression (Supplemental Fig. 2C). However, it is important a suppression of Gadd45 expression to note that although our data argue against a role for downreg- The intracellular signaling pathways through which ATM represses ulation of Gadd45a expression in DSB-induced inhibition of Rag1 gene transcription remain incompletely characterized. We previ- and Rag2 transcription in response to DSBs, our experiments do ously reported that RAG DSBs induced in primary pre-B cells not address the possibility for inactivation of Gadd45a protein signal via ATM to decrease levels of Gadd45a mRNA and pro- activity by posttranslational modifications or other mechanisms. 2950 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION Downloaded from

FIGURE 4. ATM and Nemo are required for DSBs induced in pre-B cells to downregulate Rag1 and Rag2 expression. (A) RT-PCR quantification of

Rag1, Rag2, and p21 mRNA in nonirradiated EmBCL2 pre-B cells or irradiated EmBCL2 pre-B cells at indicated times after exposure to 4 Gy of IR. Cells http://www.jimmunol.org/ were treated with 15 mM of the KU55933 ATM kinase inhibitor or vehicle (DMSO) for 48 h before irradiation or harvesting of nonirradiated cells. Data are from eight independent experiments. Data are normalized to 1.0 for nonirradiated, vehicle-treated cells within each experiment. For other conditions, data averages are shown with error bars indicating SEM. The p values were calculated by Dunnett’s posttest after ANOVA. (B) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in nonirradiated Mb1Cre+Atmflox/flox pre-B cells or irradiated Mb1Cre+Atmflox/flox pre-B cells at indicated times after exposure to 4 Gy of IR. Data are from three independent experiments. Data are normalized to 1.0 for nonirradiated cells harvested 1 h after IR exposure of irradiated samples. For other samples, data averages are shown with error bars indicating SEM. The p values were calculated by Dunnett’s posttest after ANOVA. ***p , 0.001.

DSB-induced downregulation of Rag1 and Rag2 expression purpose, we used etoposide and bleomycin because these drugs requires the NF-kB essential modulator protein are active for days in cell culture, which is the time frame over by guest on September 23, 2021 In pre-B cells, ATM activates transcription of target genes through which we can activate and monitor V(D)J recombination in pri- Tp53-mediated or Nemo-mediated intracellular signaling pathways mary pre-B cells or pre-B cell lines. We first attempted to study k (1, 22). Thus, we next investigated the roles of Tp53 and Nemo in the effect of etoposide-induced DSBs on Ig recombination in m suppressing Rag1 and Rag2 expression after the induction of DSBs primary E BCL2 pre-B cells; unfortunately, all of the etoposide in primary pre-B cells. We observed normal downregulation of Rag1 doses that we tested caused substantial apoptosis. To circumvent and Rag2 mRNAs after irradiation of primary pre-B cells cultured this obstacle, we turned to the Abelson transformed (abl) pro- from VavCre +Tp53flox/flox mice with hematopoietic lineage-specific B cell lines that we had previously used to identify RAG DSB- k deletion of Tp53 (Supplemental Fig. 2E) (32). In contrast, we de- induced feedback inhibition of Ig recombination and to study tected impairment of this DSB response in pre-B cells from Mb1Cre+ additional cellular responses to RAG DSBs (38, 40). In normal Nemoflox/floxEmBCL2 mice with B lineage–specific Nemo deletion media, Abl pro-B cells proliferate and only sporadically express k and ectopic BCL2 expression to enhance survival of Nemo-deficient Rag1 and Rag2 and recombine Ig loci (55). Addition of the pre-B cells (Fig. 5A) (33). Although the Rag1 and Rag2 mRNA STI571 Abelson kinase inhibitor causes G1 cell cycle arrest and levels at 4 h after IR were equivalent to unirradiated cells, the levels differentiation of pre-B cells that express Rag1 and Rag2 and k m at 1 h after IR were decreased as compared with unirradiated cells recombine Ig loci (55). Expression of the E BCL2 transgene (Fig. 5A). However, this downregulation of Rag1 and Rag2 mRNA enables Abl cells to survive RAG DSBs and transcribe Rag1 and in Mb1Cre+Nemoflox/floxEmBCL2 pre-B cells at 1 h after IR is to a Rag2 for several days (37). Moreover, the addition of etoposide to lesser extent than in EmBCL2 pre-B cells (Fig. 5B). Our data indicate STI571-treated, G1-arrested Abl cells induces DSBs (46). We that Nemo is required for normal DSB-induced feedback inhibition found that addition of etoposide at 24 h after STI571 addition had of Rag1 and Rag2 expression in pre-B cells, with the absence of minimal effects on G1 arrest and survival of Abl cells over the next m Nemo resulting in slower kinetics by which pre-B cells downregulate 48 h. Consistent with our data from primary E BCL2 pre-B cells m Rag1 and Rag2 expression upon irradiation. This finding suggests a (Fig. 2B), the addition of etoposide to E BCL2 Abl cells caused two-phase regulation of Rag1 and Rag2 transcription in response to downregulation of Rag1 and Rag2 mRNA and upregulation of p21 k DSBs: an early initiation phase that is partially dependent on Nemo mRNA (Fig. 6A). The small size of the J cluster enables the use k and a later maintenance phase that requires Nemo expression. of Southern blot analysis to monitor Ig recombination. In EmBCL2 Abl cells, we measure Igk recombination by quantifying DSBs induced in pre-B cells by genotoxic drugs inhibit V(D)J decreased intensity of the germline Jk band relative to the inten- k recombination of Ig loci and an integrated substrate sity of control band from a nonrearranging locus. Our Southern After demonstrating that DSBs induced by IR or genotoxic drugs blot analysis revealed that the addition of etoposide 24 h after inhibit Rag1 and Rag2 expression, we sought to test our hypoth- activating Igk recombination in EmBCL2 Abl cells inhibited fur- esis that these types of DSBs inhibit V(D)J recombination. For this ther Igk recombination over the next 48 h (Fig. 6B). We conducted The Journal of Immunology 2951

FIGURE 5. Nemo is required for pre-B cells to normally downregulate Rag1 and Rag2 expression in response to DSBs. (A)RT-PCR quantification of Rag1, Rag2, and p21 mRNA in nonirradiated Mb1Cre+Nemoflox/floxEmBCL2 pre-B cells or irradiated Mb1Cre+Nemoflox/flox EmBCL2 pre-B cells at indicated times after exposure to 4 Gy of IR. Data are from three independent experiments. The p values were calculated by Dunnett’s posttest after ANOVA. (B) RT-PCR quantification of Rag1 and Rag2 mRNA in nonirradiated or irradiated pre-B cells from EmBCL2 or Mb1Cre+Nemoflox/flox EmBCL2 mice at 1 h after exposure to 4 Gy of IR. Data are from three independent ex- periments. The p value was calculated by t test. (A and B) Data are normalized to 1.0 for nonirradiated cells harvested 1 h after IR Downloaded from exposure of irradiated samples. For other samples, data averages are shown with error bars indicating SEM. *p , 0.05. http://www.jimmunol.org/ the same experiment with EmBCL2 pre-B cell lines lacking the STI571 addition to EmBCL2-pINV Abl cells resulted in lower Artemis NHEJ factor, which is required for coding join formation levels of Rag1 and Rag2 mRNA and higher levels of p21 mRNA (56). The addition of etoposide to Artemis2/2EmBCL2 Abl cells after an additional 48 h of culture (Fig. 6E). Reflecting this sup- caused downregulation of Rag1 and Rag2 mRNA and upregula- pression of Rag1 and Rag2 expression, flow cytometry analysis tion of p21 mRNA (Fig. 6C). In Artemis2/2EmBCL2 Abl cells, we revealed that etoposide suppressed generation of GFP+ cells measure RAG cleavage of Igk loci by quantifying the relative (Fig. 6F). These data indicate that DSB-induced feedback inhi- intensity of germline Jk bands and Jk coding end bands (57). We bition of V(D)J recombination in pre-B cells occurs independent found that addition of etoposide 24 h after activating RAG of Igk-specific trans-factors. by guest on September 23, 2021 cleavage of Igk loci in Artemis2/2EmBCL2 Abl cells blocked further RAG cleavage of Igk loci over the next 48 h (Fig. 6D). We Discussion repeated this same experiment by substituting bleomycin for Our study demonstrates that immature lymphocytes downregulate etoposide. We observed that the addition of bleomycin 24 h after Rag1 and Rag2 expression and inhibit V(D)J recombination in activating RAG cleavage of endogenous Igk loci in Artemis2/2 response to DNA damage. The suppression of Rag1 and Rag2 EmBCL2 Abl cells also blocked further RAG cleavage of Igk loci mRNA levels after IR is a response shared by pro-T, pro-B, and over the next 48 h (Supplemental Fig. 3). These data demonstrate pre-B cells. Where assayed, this response is dependent on the that DSB induced by etoposide or bleomycin in pre-B cells acti- ATM kinase and also triggered by etoposide or bleomycin, pro- vates DSB-induced feedback inhibition of RAG cleavage to ini- viding strong evidence that immature lymphocytes experiencing tiate Igk recombination. DSBs inhibit Rag1 and Rag2 expression. Our use of a primary The Sleckman laboratory (27) recently reported a locus-specific mouse pre-B cell culture system enabled us to elucidate that trans-factor for RAG DSB-induced feedback inhibition of Igk transcriptional repression, rather than induction of mRNA turn- recombination that functions by downregulating transcription- over, is the mechanism by which DSBs downregulate Rag1 and linked accessibility of Jk gene segments to the RAG endonucle- Rag2 expression. This inhibition is immediate, not requiring ase. In this context, the ability of etoposide to inhibit Igk protein synthesis, and causes rapid loss in protein levels of Rag1, recombination could be because of downregulation of Rag1 and but not Rag2. Because both Rag1 and Rag2 protein are required Rag2 expression and/or Jk recombinational accessibility. There- for RAG endonuclease activity (12, 49), DSBs induced in pre- fore, we thought it was important to determine whether DSB- B cell lines by etoposide inhibit recombination of endogenous Igk induced feedback inhibition in pre-B cells also suppresses loci and a chromosomally integrated artificial substrate. These recombination of RSSs inserted at another genomic location. For data build on our prior work demonstrating that RAG DSBs in- this purpose, we turned to EmBCL2 Abl cells containing one duced during Igk recombination in a primary pre-B cell signal chromosomally integrated copy of the pINV recombination sub- through ATM to downregulate levels of Rag1 and Rag2 mRNAs strate. The pINV substrate contains a constitutive promoter and a and Rag1 protein and to feedback-inhibit additional RAG cleavage downstream GFP cDNA oriented in the antisense reading frame of Igk gene segments (24). Based on our current and prior find- that is flanked by compatible RSSs (37, 58). The orientation of ings, we conclude that any type of DSB induced anywhere in the these RSSs is such that V(D)J recombination inverts the GFP genome of a pre-B cell has the potential to signal immediate cDNA, leading to GFP expression (37, 58). Because of sporadic cessation of Rag1 and Rag2 transcription, leading to rapid Rag1 and Rag2 expression during culture, a small fraction (∼10%) downregulation of Rag1 protein expression, and thereby V(D)J of EmBCL2-pINV cells expresses GFP before the addition of recombination throughout the nucleus. Considering that immature STI571. The addition of etoposide, but not vehicle, only 24 h after T cells express an order of magnitude fewer number of Rag1 2952 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. Etoposide inhibits recombination of endogenous Igk loci and an integrated substrate. (A) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in EmBCL2 Abl cells untreated or treated with STI571 or STI571 and etoposide for the indicated amounts of time. Data are from three independent experiments. (B) Representative Southern analysis and graphical quantification of Igk recombination in EmBCL2 Abl cells untreated or treated with STI571 or STI571 and etoposide for the indicated amounts of time. Data are from three independent experiments. (C) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in Artemis2/2EmBCL2 Abl cells untreated or treated with STI571 or STI571 and etoposide for the indicated amounts of time. Data are from six independent experiments. (D) Representative Southern blot analysis and graphical quantification of Jk cleavage in Artemis2/2EmBCL2 Abl cells untreated or treated with STI571 or STI571 and etoposide for the indicated amounts of time. Data are from six independent experiments. (E) RT-PCR quantification of Rag1, Rag2, and p21 mRNA in EmBCL2-pINVAbl cells treated with STI571 or STI571 and etoposide for the indicated amounts (Figure legend continues) The Journal of Immunology 2953 monomers than Rag2 monomers (59), DSB-induced downregu- negative-acting (p50) canonical NF-kB proteins helps regulate lation of Rag1 protein in immature lymphocytes would be pre- Rag1 and Rag2 transcription in pre-B cells, with increased binding dicted to be a more effective way to reduce RAG activity than of p50 in response to DSBs repressing Rag1/Rag2 transcription. downregulation of Rag2 protein. In this context, the retention of However, considering that Nemo can function independent of NF-kB Rag2 protein in thymocytes harboring RAG DSBs at TCR loci proteins (70, 71), DSBs induced in pre-B cells might repress Rag1 (60) may result from a long half-life of endogenous Rag2 protein and Rag2 expression through other Nemo-mediated signaling enabling Rag2 to persist after DSB-induced repression of Rag1/ pathways. Another alternative possibility is that Nemo-dependent Rag2 transcription and resultant loss of Rag1 protein. Whereas NF-kB activity is necessary for constitutive expression of ATM- RAG induces two DSBs at an Igk allele, the IR dose and etoposide dependent signaling factors and/or transcriptional repressors that concentration that we used in our pre-B cell experiments each target the Rag1/Rag2 locus. induce hundreds of DSBs at random genomic locations (61, 62). While we were preparing this article, the Guikema laboratory Considering that two DSBs induced by RAG during Igk recom- reported that the DDR response regulates Rag1/Rag2 expression in bination and a larger number of DSBs induced by IR or etoposide pre-B cells through ATM-FOXO1 signaling (29). They showed cause similar reduction in Rag1 and Rag2 mRNA levels, it will be that the genotoxic drug NCS caused rapid downmodulation of important to assess whether intrinsic features of the RAG proteins Rag1 and Rag2 mRNA and Rag1 protein (they did not report on or the location, proximity, or structures of the two DSBs induced Rag2 protein) in STI571-treated human BCR-ABL+ pre-B cell by RAG during Igk recombination amplify DDR signaling. lines (BV173 and SUP-B15) and mouse v-Abl transformed pro- Our data that both ATM and Nemo are required for IR to rapidly B cell lines. Consistent with data from primary mouse pre-B cells suppress Rag1 and Rag2 mRNA levels in primary pre-B cells (47), the half-life of RAG1 in BV173 cells is relatively short Downloaded from implicates a role for canonical NF-kB transcription factors. In (,60 min). NCS causes a modest decrease of RAG1 half-life in pre-B cells, DSBs induced by RAG or etoposide signal ATM- BV173 cells but does not affect levels of Rag1 protein expressed dependent and Nemo-dependent nuclear translocation and DNA in a human embryonic kidney cell line, consistent with transcriptional binding of canonical NF-kB proteins to upregulate or down- repression being the mechanism by which DNA damage down- regulate expression of numerous genes (22). The posttranslational modulates RAG1 protein. Inhibition of ATM kinase activity an-

modification of Nemo upon DSBs or other stimuli is essential for tagonized the ability of NCS to downregulate Rag1 mRNA levels http://www.jimmunol.org/ rapid cytoplasmic-to-nuclear migration of canonical NF-kB pro- in pre-B cell lines and primary pre-B cells. NCS promotes both the teins, promoting NF-kB–mediated transcriptional activation or ATM kinase–dependent loss of FOXO1 binding to ERag and the repression of target genes (3, 63–65). The canonical NF-kB pro- binding of phospho-ATM to ERag. Because FOXO1 and unac- teins consist of p50, p65, and c-Rel, which each can function as tivated ATM are frequently in close proximity, the Guikema lab- homodimers or as heterodimers with each other or with additional oratory proposes a model wherein DSBs activate ATM to release transcription factors (63–65). Unlike p65 and c-Rel, p50 lacks a FOXO1 binding from ERag, and thereby repress Rag1/Rag2 transactivation domain and forms homodimers that repress tran- transcription. Finally, they showed that inhibition of ATM kinase scription (63, 66, 67). The mechanisms by which DSBs and other activity elevated Rag1 mRNA levels in immature B cell lines and stimuli direct the formation of specific NF-kB complexes and increased sporadic recombination of a retroviral substrate in a pre- by guest on September 23, 2021 target these to particular gene promoters remain enigmatic (63– B cell line, implying that RAG DSB-induced loss of Rag1 protein 65). Several studies have investigated the role of NF-kB proteins expression inhibits V(D)J recombination. in regulating Rag1/Rag2 expression in immature B cells, reaching Our work corroborates central findings of the Guikema labo- apparently contradictory conclusions. In immortalized and trans- ratory and presents additional data that provide substantial insights formed cell lines, the expression of a dominant negative IkBa into potential mechanisms by which immature lymphocytes direct protein that sequesters canonical NF-kB proteins in the cytoplasm DSB-induced feedback inhibition of V(D)J recombination. Both either has no effect or increases sporadic Rag1/Rag2 expression studies show that DSBs induced by a genotoxic drug in pre-B cells depending on the lines analyzed (68, 69). The inhibition of ca- signal via ATM to rapidly downregulate Rag1 and Rag2 expres- nonical NF-kB activity in primary pre-B cells by expression of the sion, but we also show that this DSB response occurs independent same dominant negative IkBa protein or by Mb1Cre-mediated of new protein synthesis and involves transcriptional repression of deletion of Nemo has no effect on Rag1 and Rag2 mRNA levels, the Rag1/Rag2 locus. In addition, we demonstrate that expression even after IL-7 withdrawal (33). However, the expression of a of the Nemo protein is required for normal DSB-induced feedback different dominant negative IkBa protein in primary pre-B cells inhibition of Rag1 and Rag2 expression in pre-B cells, with the inhibits Ab-stimulated BCR signaling from inducing Rag1/Rag2 absence of Nemo possibly resulting in slower kinetics by which transcription (53). Chromatin immunoprecipitation analyses pre-B cells suppress Rag1 and Rag2 expression in response to revealed that p50, p65, and c-Rel each bind at several sites along DSBs. When considered with work of the Guikema laboratory, our the Rag1/Rag2 locus, including the B cell–specific transcriptional data are consistent with a model wherein ATM-FOXO1 and ATM- enhancer of the Rag1/Rag2 locus (ERag) and the D3 lymphoid– Nemo signaling cooperate to rapidly initiate Rag1/Rag2 tran- specific enhancer, with BCR activation increasing p65 and c-Rel scriptional repression, while ATM-Nemo signaling maintains this binding and decreasing p50 binding (53). Moreover, pre-B cells response. In this context, the release of FOXO1 might facilitate from mice with inactivation of the Nfkb1 gene that encodes p50 NF-kB binding and/or vice versa, while NF-kB binding may more exhibit higher basal and BCR-stimulated expression of Rag1 and stably inhibit Rag1/Rag2 expression, such as by persisting Rag2 (53). Collectively, our data and these prior findings support a longer after cessation of ATM kinase activity after DSB repair. model wherein a balance of positive-acting (p65/c-Rel) and As highlighted by our prior (24) and current Gadd45a data, a

of time. Data are from three independent experiments. (F) Representative flow cytometric analysis and graphical quantification of pINV recombination in EmBCL2-pINV Abl cells treated with STI571 or STI571 and etoposide for the indicated amounts of time. Data are from four independent experiments. (A– F) Data averages are shown with error bars indicating the SEM. The p values were determined by Dunnett’s posttest after ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. 2954 DNA DAMAGE SUPPRESSES V(D)J RECOMBINATION potential mechanistic link between release of FOXO1 and inhi- cleavage activity has not been reported. Considering that the bition of Rag1/Rag2 transcription should be assessed to distinguish a AMP-activated protein kinase directly phosphorylates Rag1 to cause-effect relationship versus another unlinked ATM-dependent enhance the endonuclease activity of RAG1/RAG2 complexes correlation. Considering the complexities of ATM-FOXO1 sig- (79), it is conceivable that ATM phosphorylation of Rag1 and/or naling found by the Guikema laboratory (29), and the unresolved Rag2 directly suppresses the ability of RAG to bind and/or cleave questions over the roles of Nemo and NF-kB proteins in Rag1/Rag2 DNA. Moreover, the ability of ATM to signal nuclear export of a transcription, major obstacles exist for testing these models. Further fraction of nuclear Rag2 protein in response to DSBs (46) opens characterization of the molecular requirements and associated events the possibility that ATM might regulate the subcellular location of of DSB-induced inhibition of Rag1/Rag2 expression should open Rag1 protein. Finally, the finding of the Guikema laboratory (29) paths forward. that the genotoxic drug neocarzinostatin increases the degradation Our findings that irradiation causes downregulation of Rag1 and of Rag1 protein suggests that ATM also might regulate the sta- Rag2 mRNA levels in pro-B cells, pre-B cells, and pro-T cells, but bility of Rag1. Clearly, studies are needed to investigate the po- not in total thymocytes, have important implications for mecha- tential role of ATM-dependent posttranslational modifications of nisms by which immature lymphocytes regulate V(D)J recombi- the Rag1 and Rag2 proteins in DSB-induced inhibition of V(D)J nation. One explanation for this difference between pro-T cells and recombination. total thymocytes is that pre-T cells (CD4+CD8+ thymocytes) as- The conservation of DSB-induced feedback inhibition of Rag1 and sembling TCRa genes are not able to downregulate Rag1 and Rag2 expression among different types of immature lymphocytes Rag2 expression in response to DSBs. In this scenario, our data and between humans and mice implies that this process is important. may reflect the existence of developmental stage–specific mech- As we have outlined previously, it is possible that transiently sup- Downloaded from anisms by which immature T cells regulate Rag1/Rag2 tran- pressing RAG-mediated DNA cleavage in response to RAG DSBs scription in response to DSBs. Such putative mechanisms could induced during V(D)J recombination is critical to suppress auto- involve differential expression or activity of DDR factors that immunity from allelic inclusion and leukemia/lymphoma from suppress Rag1/Rag2 transcription or dominant roles of cis-regulatory oncogenic AgR locus translocations (24, 26, 73). The ability of non- elements that drive transcription of Rag1 and Rag2 in pre-T cells RAG DSBs to inhibit RAG-mediated cleavage of an AgR locus

(72). An inability of pre-T cells to inhibit Rag1 and Rag2 ex- could be a by-product of evolutionary selection for mechanisms that http://www.jimmunol.org/ pression in response to DSBs would be unique among the types enable RAG DSBs to direct monoallelic recombination of Igk, IgH, of immature B and T cells that assemble AgR genes. Mirroring and TCRb loci. In pre-T cells, RAG (and non-RAG) DSBs might this, TCRa gene assembly in pre-T cells is not regulated to enforce signal posttranslational inhibition of RAG activity and/or repression allelic exclusion, whereas recombination of the only or predomi- of TCRa recombination potential. Alternatively, unique require- nant AgR genes assembled in pro-B cells (IgH), pre-B cells (Igk), ments for TCRa recombination may have selected for mechanisms and pre-T cells (TCRb) is controlled to occur on one allele at a that prevent DSB-induced inhibition of V(D)J recombination. Cells time (73). However, an important caveat to note is that our data that assemble AgR genes via RAG DSB intermediates also experi- cannot rule out that pre-T cells experiencing DSBs from 10 Gy of ence DSBs from other endogenous factors such as transcription and radiation do not suppress Rag1 and Rag2 transcription because cellular metabolism. Although one DSB can cause a translocation, a by guest on September 23, 2021 they are directly induced to die. We previously reported that mice second simultaneous DSB increases translocation frequencies by lacking ATM develop greater frequencies of mature lymphocytes several orders of magnitude (80). Therefore, regardless of whether expressing Igk, IgH,orTCRb genes from both alleles (24, 26). the ability of immature lymphocytes to downregulate RAG activity These findings provided evidence for RAG DSB-induced feedback in response to non-RAG DSBs is fortuitous, this cellular response inhibition of IgH, Igk, and TCRb recombination (24, 26, 57). Yet, likely helps suppress oncogenic AgR locus translocations. as we noted, the slower kinetics of coding join formation in cells lacking ATM could cause this increased allelic inclusion, rather Disclosures than feedback inhibition of V(D)J recombination through tran- C.H.B. is an expert witness on retainer for Regeneron Inc. The other authors scriptional repression of Rag1/Rag2 (26). Our current data show have no financial conflicts of interest. that DSBs induced in pro-B, pre-B, and pro-T cells indeed suppress Rag1 and Rag2 expression, providing additional support for our model wherein the responses of these immature lymphocytes to References 1. Shiloh, Y., and Y. Ziv. 2013. The ATM protein kinase: regulating the cellular RAG DSBs is one essential mechanism for enforcing AgR allelic response to genotoxic stress, and more. Nat. Rev. Mol. Cell Biol. 14: 197–210. exclusion (26). The additional essential mechanisms likely include 2. Helmink, B. A., and B. P. Sleckman. 2012. 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