ATM-Dependent Phosphorylation of Nemo SQ Motifs is Dispensable for Nemo-Mediated Gene Expression Changes in Response to DNA Double Strand Breaks

Abstract

In response to DNA double strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm where it promotes monoubiquitylation of Nemo and resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as lipopolysaccharides (LPS). To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy, fertile, and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

Introduction

DNA damage is a major risk to cellular survival and genomic integrity, yet, it is constantly produced by cell intrinsic mechanisms. DNA damage occurs either intentionally during normal processes, such as by topoisomerases during DNA replication or transcription to reduce torsional stress, or inadvertently due to DNA replication hazards or metabolic by-products. DNA double strand breaks (DSBs) are amongst the most threatening genomic lesions because, if not properly repaired, they can trigger cell death or create genomic lesions that drive cellular transformation (1). Consequently, cells have evolved a rapid and efficient DNA damage response that coordinates DSB repair with cellular proliferation and survival to maintain genomic integrity and suppress malignant transformation (2). This DNA damage response is initiated by sensor proteins that recognize DSBs in chromatin and direct repair through homologous recombination or non-homologous end joining (NHEJ) pathways depending on when DSBs arise during the cell cycle (2, 3). Sensor protein complexes bound at DNA damage sites stimulate a complex signaling cascade in large part via activation of three related phosphoinositide 3-kinase-like serine-threonine protein kinases: Ataxia Telangiectasia Mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ataxia telangiectasia and RAD3 related (ATR) (4). Once activated, these kinases phosphorylate their protein targets at serine or threonine residues preceding glutamine residues (S/T-Q motifs) to execute the DNA damage response (5). These protein targets include additional kinases, such as CHK1 and CHK2, which also phosphorylate numerous substrates in the DNA damage response (4). Together, these DNA damage response kinases induce post translational modifications of numerous proteins to alter their activities and stimulate transcriptional programs, thereby activating cell cycle checkpoints and factors that determine cellular fate. For example, ATM phosphorylates and stabilizes the transcription factor p53, which induces expression of proteins that trigger cell cycle checkpoints and apoptosis if DNA damage is too severe (5). Simultaneously, ATM also activates NF-κB transcription factors to upregulate transcription of anti-apoptotic genes and thus promote cell survival, giving cells more time to repair DNA damage (6).

The ATM-dependent activation of NF-κB transcription factors in response to DSBs employs nuclear-to-cytoplasmic signaling that integrates into canonical signaling cascades induced by extracellular factors, such as pro-inflammatory lipopolysaccharides (LPS). In the canonical pathway of NF-κB, external stimuli activate the Inhibitor of κB kinase (IKK) complex composed of the IKKα and IKKβ kinases and the IKKγ regulatory subunit, which is known also as the NF-κB essential modulator (Nemo) protein (7, 8). Once activated, IKKβ phosphorylates Inhibitor of κB (IκB) proteins to trigger their degradation. As IκB proteins sequester NF-κB transcription factors in the cytoplasm, IκB destruction enables NF-κB transcription factors to move into the nucleus and modulate transcription of their target genes. Studies in cell lines have elucidated that ATM can activate NF-κB factors by two distinct signaling pathways that converge upon and activate the IKK complex. In response to DSBs, the poly(ADP-ribose) polymerase-1 DNA damage sensor facilitates the formation of a nuclear signaling complex containing ATM, Nemo, and a SUMO E3 ligase protein (9, 10). The formation of this complex leads to accumulation of sumoylated Nemo protein, which ATM can phosphorylate at serine 85 of an SQ motif (10–12). This ATM-mediated phosphorylation permits monoubiquitylation of Nemo, which promotes nuclear export of Nemo:ATM complexes to activate cytoplasmic IKK (12). Additionally, DSBs also stimulate Nemo-independent movement of ATM into the cytoplasm where ATM targets upstream modulators of IKK that trigger monoubiquitylation of Nemo and resulting IKK activation (13). The experiments that elucidated each of these two distinct ATM- and Nemo-dependent intracellular signaling cascades were conducted using different immortalized/transformed cell lines and neither of the studies addressed both pathways. Accordingly, the in vivo relevance and potential interdependence or redundancy of these two distinct ATM- and Nemo-dependent mechanisms for signaling NF-κB-mediated changes of gene expression in response to DSBs remain undetermined.

Although hazardous, DSBs are essential for biology. A prime example of this is the requirement for DSBs in lymphocyte-specific genomic rearrangements that establish antigen receptor (AgR) gene diversity (14). Germline immunoglobulin (Ig) and T cell receptor (TCR) AgR gene loci consist of variable (V), joining (J), and sometimes diversity (D) gene segments located upstream of constant (C) region exons. In developing B and T cells, the lymphocyte-specific RAG1/RAG2 (RAG) endonuclease induces DSBs adjacent to a pair of gene segments and functions with the NHEJ pathway to resolve these DSBs into the V(D)J rearrangements that create variable region exons of AgR genes (15, 16). The assembled variable region exons and downstream C region exons comprise a complete AgR gene. Together, the combinations of V(D)J rearrangements and imprecision of NHEJ-mediated DSB repair generate the vast numbers of diverse Ig and TCR genes necessary for effective adaptive immunity. The importance of V(D)J recombination and necessary RAG DSBs intermediates is evident from RAG1 or RAG2 mutations that decrease RAG endonuclease activity ablating B and T cell development, thereby causing severe combined immunodeficiency (17). Notably, RAG DSBs function beyond obligate intermediates for AgR gene assembly. They activate the ATM-mediated DNA damage response to promote efficient repair and suppress aberrant resolution as oncogenic lesions that predispose lymphoid cancers (18–20). ATM activated by RAG DSBs also signals via Nemo to mediate changes in expression of lymphocyte-specific genes that regulate lymphocyte specific functions (21–23). Notably, DSBs induced within developing B cells by genotoxic agents such as etoposide trigger the same ATM-dependent/Nemo-independent and ATM/Nemo-dependent gene expression changes (21, 24), implying that lymphocytes may have evolved cell type-specific DSB response signals to facilitate adaptive immunity.

We have a long-standing interest in elucidating how DSBs induced by RAG during V(D)J recombination alter gene expression and in determining how these mechanisms regulate B and T cell development. One emphasis of our work has been on RAG DSB-signaled transient feedback inhibition of V(D)J recombination through repression of Rag1 and Rag2 transcription, which we have shown depends on both ATM and Nemo (22, 23). A challenge that we have encountered is the pleotropic consequences of inactivating ATM or Nemo, even when done so specifically in developing B or T cells (22, 23, 25). To overcome this obstacle, we sought to specifically prevent ATM-mediated activation of Nemo following induction of DSBs in a manner that would allow us to study the role of DSB-induced NF-κB activation in developing B and T lymphocytes in vivo. In a Nemo-deficient mouse pre-B cell line, the ectopic expression of human NEMO protein with serine 85 of an SQ motif replaced by alanine prevents both ATM-mediated phosphorylation of NEMO protein at this site and activation of NF-κB in response to etoposide-induced DSBs (12). Notably, this NEMO^S85A phosphomutant supported normal activation of NF-κB downstream of LPS, demonstrating a specific requirement of serine 85 for regulating NF-κB factors following DSBs (12). Therefore, we employed CRISPR/Cas9 genome editing to generate mice expressing endogenous Nemo protein carrying serine 85 mutated to alanine (S85A) for this serine, and its proceeding glutamine, are conserved between mouse and human proteins. In mice, germline inactivation of Nemo causes embryonic lethality, while conditional Nemo inactivation within lineage-committed lymphocytes reduces numbers of developing and mature B and T cells including Igλ⁺ B cells (26–30). We show that our Nemo^S85A mice are viable, fertile, healthy, present no obvious phenotypes, and exhibit overall normal development and numbers of mature B and T cells including Igλ⁺ B cells. Unexpectedly, we found that primary bone marrow B lineage cells of Nemo^S85A mice supported normal Nemo-dependent changes in the expression of Rag1, Rag2, and validated NF-κB responsive genes following exposure to etoposide. To account for potential compensatory ATM-dependent phosphorylation of Nemo at its two additional SQ motifs, we employed CRISPR/Cas9 genome editing in Nemo^S85A zygotes to create mice carrying serine to alanine mutation of all three Nemo SQ motifs. We observed identical normal phenotypes in these mice and their primary B lineage cells as for Nemo^S85A mice. Moreover, we show that LPS treatment of primary B lineage cells from these mice or Nemo^S85A mice induces normal increases in expression of all Nemo-dependent NF-κB target genes assayed. Therefore, we conclude that ATM-mediated phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes, at least in primary mouse B lineage cells.

Materials and Methods

Mice

All experimental mice assayed in this study were between 4 and 6 weeks old, of mixed sex, and housed under specific pathogen free conditions at the Children’s Hospital of Philadelphia (CHOP). Animal husbandry, breeding, and experiments were performed in accordance with national guidelines and regulations and approved by the CHOP Institutional Animal Care and Use Committee. Experimental mice were euthanized by exposure to CO2, followed by cervical dislocation. We used CRISPR/Cas9 genome editing in C57BL/6 zygotes to generate mice carrying the Nemo serine 85 to alanine replacement by mutating the Nemo allele (Nemo^SA). To generate the Nemo^SA allele, we used the online CRISPOR tool (http://crispor.tefor.net/; (31)) to identify an optimal CRISPR RNA (crRNA) sequence closest to the Nemo serine 85 codon. From this, we designed a single guide RNA (sgRNA) template that fused the identified crRNA sequence and the trans activating crRNA (tracrRNA) sequence into a single oligo, the Nemo S85A sgRNA Template, that we ordered from Integrated DNA Technologies (Supplemental Table 1) (32). We generated the Nemo S85A sgRNA from this template DNA using T7 promoter overlapping PCR and in vitro transcription (33, 34). Briefly, to generate the Nemo S85A sgRNA from this template DNA, we first PCR amplified the template with a forward primer that contained a T7 promoter and the common sgRNA reverse primer (sequences found in Supplemental Table 1) using Phusion High Fidelity DNA Polymerase (NEB, Cat. No. M0530S). We set up eight PCR reactions to amplify enough template DNA with the following final concentrations of each component that differed from the manufacturer’s recommendations: 1.5mM MgCL₂, 12ng/μL sgRNA Template, 0.8 μM T7 sgRNA F primer, 0.8 μM sgRNA R primer, and 0.6units Polymerase/50μL PCR reaction. The thermocycler setting consisted of 94 °C for 2 min, 35 cycles of 94 °C for 20 s, 55 °C for 30 s, and 72 °C for 40 s, with a final stage of 72 °C for 7 min. Once amplified, we consolidated reactions and purified the template using a QIAquick PCR purification Kit (Qiagen, Cat. No. 28104) according to manufacturer’s instructions. We measured the concentration of the product, diluted it to 125ng/μL, and confirmed correct band size (125bp) by gel electrophoresis. Next, we generated the Nemo S85A sgRNA by in vitro transcribing the sgRNA template PCR product using the MEGAshortscript Kit (Thermo Fisher, Cat. No. AM1354) according to the manufacturer’s instructions, including the DNase treatment. We purified the in vitro transcribed sgRNA using a MegaClear Kit (Thermo Fisher, Cat. No. AM1908) according to manufacturer’s instructions and verified correct size by gel electrophoresis. Next, we prepared the microinjection cocktail for the CHOP Transgenic Core who injected C57BL/6 zygotes with a mixture of Nemo S85A sgRNA, Cas9 protein (QB3 Macrolab, University of California at Berkeley), and the Nemo S85A single stranded homology directed repair (HDR) oligo (Integrated DNA Technologies; sequence in Supplemental Table 1) (33, 35). Founder mice containing the Nemo^SA mutation were identified by PCR of tail DNA using the NemoSeq F and Nemo S85A R primers (Integrated DNA Technologies; Supplemental Table 1). The Nemo^SA mutation was sequence verified by PCR of tail DNA from F1 Nemo^SA male mice using NemoSeq F and NemoSeqR primers (Integrated DNA Technologies; Supplemental Table 1). The prepared sequencing reaction was submitted to the University of Pennsylvania Genomic and Sequencing Core for Sanger Sequencing. Subsequent genotyping was done using the NemoSeq F and either the Nemo S85 WT R or Nemo S85A R primer to identify the wild-type (WT) and Nemo^SA allele, respectively (Integrated DNA Technologies; Supplemental Table 1).

To generate mice carrying the Nemo serine 116 and 156 to alanine replacements, we used Easi-CRISPR genome editing as described (36). Briefly, we used the CRISPOR tool to identify optimal crRNA sequences closest to the Nemo serine 116 and 156 codons (31) and ordered Alt-R^® CRISPR-Cas9 crRNAs from Integrated DNA Technologies containing the associated guide RNA (gRNA) sequences for each mutation (Supplemental Table 1). To generate mutations using CRISPR/Cas9, The CHOP transgenic core electroporates zygotes with a mixture of crRNA, tracrRNA, Cas9 protein, and single-stranded oligonucleotides as described (36). To generate mice with Nemo serine 85, 116, and 156 replaced with alanine, the CHOP transgenic core sequentially electroporated zygotes generated from mating superovulated Nemo^SA/SA females with C57BL/6 males as these zygotes would have an allele already containing the Nemo^S85A mutation. First, they electroporated the zygotes with a mixture containing the NemoS116A crRNA, tracrRNA, Cas9, and NemoS116A HDR oligo (Integrated DNA Technologies; Supplemental Table 1). The following day, the zygotes were electroporated again with a mixture containing the NemoS156A crRNA, tracrRNA, Cas9, and NemoS156A HDR oligo (Integrated DNA Technologies; Supplemental Table 1). Founder mice were screened for all three mutations by PCR of tail DNA using the NemoSeq F and NemoS85A R or NemoS116A R and NemoS156 Seq F and NemoS156A R primers (Integrated DNA Technologies; Supplemental Table 1). F1 male mice showing evidence of all three mutations were used to sequence verify the Nemo^S85A,S116A and Nemo^S156A mutation by PCR of tail DNA using NemoSeq F and NemoSeq R, and NemoS156 Seq F and NemoS156 Seq R primers, respectively (Integrated DNA Technologies; Supplemental Table 1). The latter reaction and sequencing identified the three-nucleotide deletion that results in deletion of Nemo threonine 164, allowing confirmation of an allele with Nemo serine 85, 116, and 156 replaced with alanine and threonine 164 deleted (Nemo^3SAΔT allele). Subsequent genotyping for Nemo^S116A was done using the NemoSeq F and either the Nemo S116 WT R or Nemo S116A R primer to identify the WT and Nemo^S116A mutation, respectively (Integrated DNA Technologies; Supplemental Table 1). Subsequent genotyping for Nemo^S156A was done using the NemoS156 Seq F and either the Nemo S156 WT R or Nemo S156A R primer to identify the WT and Nemo^S156A mutation, respectively (Integrated DNA Technologies; Supplemental Table 1). The Nemo^SA and Nemo^3SAΔT alleles were bred with C57BL/6 mice for two generations before crossing males and females containing the mutation together to generate experimental mice. The wild-type control mice were C57BL/6 mice. The EμBCL2 and Mb1Cre:EμBCL2:Nemo^flox (24) mice were on a mixed 129S1/SvImJ and C57BL/6 background.

Flow Cytometry

Single cell suspensions were generated from the bone marrow, thymuses, and spleens of mice, processed, and stained as described (23). To evaluate B cell development in the bone marrow and spleen, 10 million cells were stained with BUV395 Rat anti-mouse CD45R/B220 (563793, Clone RA3–6B2; BD Biosciences), APC Rat anti-mouse CD43 (560663, Clone S7; BD Biosciences), BV786 Rat anti-mouse IgM (743328, Clone II/41, BD Biosciences), PE Rat anti-mouse Ig κ light chain (559940, Clone 187.1, BD Biosciences), and FITC Rat anti-mouse Ig, λ1, λ2, & λ3 light chain (553434, Clone R26–46, BD Biosciences). Pro-B cells were gated on lymphocytes, single, live, B220^loCD43^lo, and IgM⁻ cells. Pre-B cells were gated on lymphocytes, single, live, B220^loCD43⁻, and IgM⁻ cells. Immature B cells were gated on lymphocytes, single, live, B220^loCD43⁻, IgM⁺ cells and then Igκ⁺ or Igλ⁺, to identify Immature B Igκ⁺ or Igλ⁺ cells, respectively. Mature B cells in the spleen were gated on lymphocytes, single, live, CD43⁻, and B220⁺IgM⁺ cells and then Igκ⁺ or Igλ⁺, to identify Mature B Igκ⁺ or Igλ⁺ cells, respectively. To evaluate the frequency of thymocytes in each double negative (DN) stage in the thymus, 10 million thymocytes were stained with V450 Rat anti-mouse CD4 (560468, Clone RM 4–5, BD Biosciences), APC Rat anti-mouse CD8α (100712, Clone 63–6.7, BioLengend), FITC Rat anti-mouse CD44 (553133, Clone IM7, BD Biosciences), PE-Cy7 Rat anti-mouse CD25 (552880, Clone PC61, BD Biosciences), and a lineage (Lin) panel composed of PE Hamster anti-mouse TCRβ (12–5961-82, Clone H57–597, Thermo Fisher), PE Hamster anti-mouse TCRδ (553178, Clone GL3, BD Biosciences), PE Rat anti-mouse CD45R/B220 (553089, Clone RA3–6B2, BD Biosciences), PE Rat anti-mouse CD19 (553786, Clone 1D3, BD Biosciences), PE Rat anti-mouse CD11b (553311, Clone M1/70, BD Biosciences), PE Hamster anti-mouse CD11c (557401, Clone HL3, BD Biosciences), PE Mouse anti-mouse NK1.1 (553165, Clone PK136, BD Biosciences), and PE anti-mouse Ter119 (553673, Clone TER-119, BD Biosciences). DN cells were gated on lymphocytes, single, live, CD4⁻CD8⁻, Lin⁻ cells and then separated into stages by CD44 and CD25. To analyze T cell development in the thymus and spleen, 10 million cells were stained with APC-eFluor^™ 780 Rat anti-mouse CD4 (47–0042-82, Clone RM4–5, Thermo Fisher), Pacific Blue^™ Rat anti-mouse CD8α (558106, Clone 5–6.7, BD Biosciences), APC Hamster anti-mouse TCRβ (553174, Clone H57–597, BD Biosciences), and PE Hamster anti-mouse TCRδ (553178, Clone GL3, BD Biosciences). Thymocyte developmental stage (CD4⁻CD8⁻ DN, CD4⁺CD8⁺ double-positive (DP), CD4⁺ single positive (SP), or CD8⁺ SP) cells were gated on lymphocytes, single, and live cells. The αβ or γδ T cells were gated on lymphocytes, single, live, and either TCRβ⁺ or TCRδ⁺ cells, respectively. In the spleen, αβ T cells were then gated on CD4⁺ or CD8⁺ cells to identify mature SP αβ T cells. Data were collected on an LSRFortessa and analyzed with FlowJo software (Tree Star).

Culturing Total Bone Marrow

Total bone marrow cultures were completed as previously described (23). Briefly, total bone marrow was flushed from all leg bones of individual mice of the appropriate genotype, depleted of red blood cells, pelleted by centrifugation, and resuspended in IL7⁻ media (RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 13 nonessential amino acids, 1 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin-streptomycin, 50 mM 2-ME). The cells were split into 5 aliquots, one was collected as a baseline sample and the others were cultured for 1 or 4 hours after being treated with DMSO or 10 μg/mL of etoposide (Sigma-Aldrich, E2600000). The cells were collected in 1mL of TRIzol for RNA analysis.

Ex vivo Primary Pre-B Cell Cultures

Ex vivo primary pre-B cell cultures were conducted as previously described (23). Briefly, primary bone marrow was harvested by flushing all leg bones of at least two mice of the appropriate genotype for each culture. These bone marrow cells were cultured for 4 days in IL7⁺ media, which is IL7⁻ media supplemented with 5 ng/ml IL7 (R&D Systems, 407-ML). Cells were cultured in IL7 at a density of 5 million cells per milliliter of media. To induce G1 arrest and activate transcription of Rag1 and Rag2 and Igk recombination by IL7 withdrawal, we pelleted cells by centrifugation, resuspended them in IL7⁻ media at a density of 2 million cells per milliliter. After 48 hours in IL7⁻ media, cells were aliquoted into 24 well plates to be treated with vehicle control (DMSO), 15 μM ATM inhibitor KU55933 (Sigma-Aldrich, SML1109), or 10 nM IKKβ inhibitor TPCA-1 (SelleckChem, S2824) for two hours as a pre-treatment before the cells were then either treated with additional DMSO or 10 μg/mL etoposide (Sigma-Aldrich, E2600000). The cells were collected four hours after etoposide treatment in 1mL of TRIzol (Life Technologies, 15596018) for RNA analysis. In these same experiments, 48-hour IL7 withdrawn cells were also aliquoted to treat cells with either vehicle control or 15 μg/mL of lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391–1MG). These cells were collected four hours after LPS treatment in 1mL of TRIzol (Life Technologies, 15596018) for RNA analysis.

Isolating CD19⁺ Splenocytes

Single-cell suspensions were generated from the spleens of individual mice of the appropriate genotype and depleted of RBCs. The EasySep^™ Mouse CD19 Positive Selection Kit II (STEMCELL Cat. No. 18954) was used to select CD19⁺ cells according to the manufacturer’s recommendations. Once isolated, the CD19⁺ splenocytes were resuspended to ~2million cells per milliliter in IL7⁻ media and ~2million cells were treated with DMSO, 15 μg/mL of lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391–1MG), or 10 μg/mL etoposide (Sigma-Aldrich, E2600000). The cells were collected after four hours of treatment in 1 mL or 250 μL of Trizol (Life Technologies, 15596018) for qRT-PCR or RNAseq analysis, respectively.

Real-time PCR quantification of mRNA

RNA was isolated and processed as described (23). Briefly, total RNA was isolated using the RNeasy mini kit (Qiagen, 74106), treated with DNase (RNase-Free DNase Set, Qiagen, 79254), and reverse transcribed to generate cDNA with High-Capacity RNA-to-cDNA^™ Kit (Applied Biosystems, 4387406) according to manufacturer’s directions. The cDNAs were then used as a template for real-time PCRs (RT-PCRs) performed with Power SYBR Green Master Mix (Applied Biosystems, 4367659) and run on a Quant Studio Flex 7 machine using the primers in Supplemental Table 1 for each corresponding transcript. Values were calculated by ΔΔCt analysis by first normalizing to Cd19 as indicated, and then the indicated sample within each experiment.

Western Blot

Total bone marrow cells were flushed from all of the leg bones of individual mice of the appropriate genotype and depleted of RBCs. Cells were pelleted by centrifugation and flash frozen for further processing. Cells were lysed using RIPA Buffer supplemented with Protease/Phosphotase Inhibitor Cocktail (Cell Signaling Technology, 5872S) and Benzonase Nuclease (Sigma-Aldrich, E1014–5KU). Protein in each sample was quantified using the Pierce^™ BCA Protein Assay Kit (Thermo Fisher, 23227) according to the manufacturer’s recommendations. Western Blot samples were prepared using NuPAGE LDS Sample Buffer (Thermo Fisher, NP0007) and subjected to polyacrylamide gel electrophoresis on NuPAGE^™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gels (Thermo Fisher, NP0322) and transferred to Invitrolon^™ PVDF/Filter Paper Sandwiches, 0.45 μm (Thermo Fisher, LC2005). Blocking was performed in TBST containing 5% non-fat dry milk for each antibody. Antibodies used were Recombinant Anti-IKK gamma/NEMO antibody (Abcam, ab178872) and Lamin A/C (4C11) Mouse mAb (Cell Signaling Technology, #4777). Blots were washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies. The blots were visualized using WesternBright ECL HRP Substrate (Advansta, K-12045-C20) on the GBox Chemi XRQ (Syngene).

RNA Sequencing

RNA extraction, RNA library preparations, sequencing reactions and bioinformatic analysis were conducted at GENEWIZ/Azenta. (South Plainfield, NJ, USA) as follows: Total RNA was extracted from frozen cell samples using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). ERCC Ex-fold RNA reagent (Cat: 4456739) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer’s protocol. Strand-specific RNA sequencing libraries was prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, the enriched RNAs were fragmented for 8 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3’ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on the flowcell. After clustering, the flowcell was loaded on the Illumina instrument according to manufacturer’s instructions. The samples were sequenced using a 2×150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and demultiplexed using Illumina’s bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.

RNA Sequencing Analysis

The RNA-seq fastq files were analyzed using the snakemake pipeline: https://github.com/khayer/rna_seq_standard_pipeline. In brief, raw RNA-seq reads were trimmed using bbduk as part of bbmap (version 38.92: https://sourceforge.net/projects/bbmap/). The trimmed reads were then aligned to mm10 using STAR (version 2.7.9a) (37). Next, bamCoverage from the deeptools package (version 3.5.1) was employed to generate bigWig coverage files, applying flags to differentiate coverage tracks by strand and to normalize using counts per million (cpm) (38). The normalized bigWig coverage files were then uploaded to the UCSC genome browser (39).

Gene expression levels were quantified using TPMcalculator (version 0.0.3) with the GENCODE annotation for the mm10 genome (version M23_GRCm38.p6) (40). For normalization and identification of significantly differentially expressed genes, the raw read counts were analyzed with the limma-voom package from Bioconductor (version 3.54.2) (41). From this data, we defined three subsets of genes. The first subset was etoposide-dependent genes, which had a log₂(Fold-Change Etoposide/DMSO) > 1 or < −1 and a p-value < 0.05 when comparing etoposide and DMSO treated CD19⁺ splenocytes from WT mice. Within the etoposide-dependent genes that were expressed above the 20^th quantile in average expression, we defined the Nemo-dependent ones as genes with a log₂(Fold-Change Etoposide/DMSO) < 1 or > −1 in Nemo⁻ CD19⁺ splenocytes and a p-value < 0.05 when comparing the log₂(Fold-Change Etoposide/DMSO) between WT and Nemo⁻ splenocytes. We introduced the qualification that the genes be expressed above the 20th quantile of average expression in this group to ensure observed changes in fold-change values were not an artifact of low expression. Finally, within the etoposide and Nemo-dependent genes, we defined a subset of genes as Nemo^3SAΔT-dependent, meaning when the log₂(Fold-Change Etoposide/DMSO) between WT and Nemo^3SAΔT samples were compares, the p-value < 0.05. The complete list of each of these subsets of genes can be found in Supplemental Table 2. To visually depict expression patterns of these subsets of genes in WT CD19⁺ splenocytes relative to either Nemo⁻ or Nemo^3SAΔT CD19⁺ splenocytes, scatterplots of the log₂(Fold-Change Etoposide/DMSO) values were plotted using the R package ggplot2 (42).

Data Availability

The data in Figure 6 is openly available in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE264315.

FIGURE 6.

Loss of Nemo serine 85, 116, 156, and threonine 164 does not impact global gene expression changes induced by DSBs in primary CD19⁺ splenocytes like complete loss of Nemo. (A-B) qRT-PCR quantification of Nfkbia, Nfkbie, Nfkb2, or RelB transcripts in primary CD19⁺ splenocytes from Nemo⁺(WT), Nemo^SA, Nemo^3SAΔT, and Nemo⁻ mice. CD19⁺ splenocytes were treated with either DMSO, (A) lipopolysaccharide (LPS; 15μg/mL in media), or (B) etoposide (10 μg/mL in DMSO) and collected 4 hours later. Individual points represent replicates (WT, n=6; Nemo^SA, n=6; Nemo^3SAΔT, n=5; Nemo⁻, n=4) from two independent experiments. Bars represent the mean ± SEM. Each transcript was first normalized to Cd19 to account for cDNA input and then normalized to the DMSO treated cells within each genotype, making this value 1, which is represented by the dotted line in each graph. Statistical significance determined by Two-way ANOVA and Šídák’s multiple comparisons test comparing each value to the WT LPS or etoposide treated sample (*, <0.0332; **, <0.0021; ***, <0.0002; ****, <0.0001). (C-D) Scatterplots depicting the log₂(Fold-Change Etoposide/DMSO) in gene expression as measured by RNASeq for etoposide-dependent genes expressed above the 20^th percentile in average expression in (C) Nemo⁻ vs WT, or (D) Nemo^3SAΔT vs WT CD19⁺ splenocytes. CD19⁺ splenocytes of WT, Nemo^3SAΔT, and Nemo⁻ mice were treated with DMSO or etoposide (10 μg/mL in DMSO) for four hours and collected for RNASeq (n=3 for each genotype from one independent experiment). Etoposide-dependent genes are defined as genes having log₂(Fold-Change Etoposide/DMSO)) > 1 or < −1 and p < 0.05 in WT CD19⁺ splenocytes and each gene is represented by a light grey dot. The dots outlined in dark grey represent the etoposide-dependent genes that are also Nemo-dependent, defined as having a log₂(Fold-Change Etoposide/DMSO)) > −1 and < 1 in Nemo⁻ CD19⁺ splenocytes and p < 0.05 when comparing the log₂(Fold-Change Etoposide/DMSO) in WT and Nemo⁻ CD19⁺ splenocytes. The two dots with a dark black X represent Nemo-dependent genes that have a p < 0.05 when comparing the log₂(Fold-Change Etoposide/DMSO) in WT and Nemo^3SAΔT CD19⁺ splenocytes. When the log₂(Fold-Change Etoposide/DMSO) values for a gene are near a line with a slope of 1, this indicates that the etoposide-induced fold-change for the gene is similar between the two genotypes being compared on one scatterplot.

Statistical Analyses

Statistical analyses were completed with Prism 9.

Results

Nemo serine 85 is dispensable for normal B and T cell development and DSB-induced repression of Rag1 and Rag2 and upregulation of Nfkbia in primary B lineage cells.

To elucidate in vivo mechanisms and roles of ATM-dependent changes in expression of NF-κB-regulated genes, we sought to specifically block the activation of endogenous Nemo protein by ATM following the induction of DSBs. In a Nemo-deficient mouse pre-B cell line, the expression of a human NEMO protein with serine 85 of an SQ motif replaced by alanine blocks ATM-mediated phosphorylation of NEMO at this site and abrogates NF-κB activation following treatment with the genotoxic agent etoposide but not pro-inflammatory LPS (12). The serine 85 of human NEMO protein is conserved in mouse Nemo protein, with each followed by glutamine (Fig. S1 A, B). Therefore, we employed CRISPR/Cas9 genome editing in C57BL/6 mouse zygotes to create mice with a Nemo gene mutation that encodes Nemo protein carrying a serine 85 to alanine replacement, which we refer to as the Nemo^SA phosphomutant. By PCR analysis of tail DNA, we detected evidence of the desired Nemo^SA gene mutation in several founder mice. We bred these founders with otherwise wild-type (WT) C57BL/6 mice and PCR/sequenced tail DNA of F1 mice to verify the desired Nemo^SA gene mutation and no changes in flanking nucleotide sequences (Fig. S1 C). We selected one sequence-verified founder line and bred the Nemo^SA allele onto the C57BL/6 background for at least one generation before breeding together males and females harboring the X-linked Nemo^SA allele to generate mice that expressed Nemo^SA protein from one (male) or two (female) Nemo^SA alleles, hereafter referred to as Nemo^SA mice regardless of sex. The analysis of mice from the F2 or subsequent generations reduced the likelihood of mice having off-target CRISPR/Cas9-induced mutations that could influence phenotypes. In mice, complete inactivation of Nemo causes embryonic lethality (26). However, we found that Nemo^SA mice regardless of sex were born at a normal frequency, viable, fertile, and displayed no visual phenotypes (data not shown). These findings alone indicate that expression of only endogenous Nemo^SA protein does not have the severe consequences of complete absence of Nemo protein.

We next evaluated the potential consequences that expression of only endogenous Nemo^SA protein exerts upon B and T lineage lymphocytes and their development. Male embryonic stem cells with inactivation of the Nemo gene cannot produce mature B and T cells when used to create chimeric mice (26). Moreover, conditional inactivation of Nemo in lineage-committed B or T lymphocytes established that cell-intrinsic Nemo expression is required for normal development and maintenance of mature B and T cells (27–30). Conditional inactivation of Nemo initiating at the earliest stage of B cell development has no effect on B cell development from the pro-B stage through the immature B cell stage, but reduces the number of immature Igλ⁺ B cells and more so the number of mature splenic B cells (28). Conditional inactivation of Nemo initiating at the CD4⁺CD8⁺ double-positive (DP) thymocyte stage of αβ T cell development decreases numbers of CD4⁺ or CD8⁺ single-positive (SP) thymocytes and mature splenic αβ T cells (30). We employed flow cytometry and cell counting to interrogate B and T cell development of Nemo^SA and WT control mice. We observed no differences in the frequencies or numbers of pro-B, pre-B, or immature B cell populations, including immature Igκ⁺ and Igλ⁺ B cells, in the bone marrow of Nemo^SA as compared to WT mice (Fig. 1 A, B). Moreover, the frequency and total number of mature splenic IgM⁺, Igκ⁺, or Igλ⁺ B cell populations were equivalent among Nemo^SA and WT mice (Fig. S2 A, B). We detected no differences of frequencies or numbers of cells at each distinct thymocyte developmental stage (CD4⁻CD8⁻ double-negative (DN), DP, CD4⁺ SP, or CD8⁺ SP) or αβ or γδ T cells among Nemo^SA and WT mice (Fig. 1 C–F). Furthermore, we found equivalent frequencies and numbers of mature splenic αβ and γδ Τ cells between these genotypes (Fig. S2 B, C). Collectively, these observations show that expression of only endogenous Nemo^SA protein has no obvious effects on the overall development or maintenance of mature B and T cell populations. In this regard, the integrity of Nemo serine 85 is dispensable for the development and homeostasis of B and T lineage lymphocytes.

FIGURE 1.

Nemo serine 85 is dispensable for normal overall B and T cell development. (A) Representative flow plots analyzing the frequency of cells at indicated B cell developmental stages in the bone marrow of WT or Nemo^SA mice. The bold gates indicate the population shown in the next panel. The first panel presents live lymphocytes that express B220 or CD43. The second panel shows B220^loCD43⁻ cells that express IgM. The third panel shows IgM⁺ cells that express Igκ or Igλ. The frequencies are shown in the flow plots if those are the values quantified. The frequencies are not shown if the values graphed incorporate prior gating frequencies. (B) Quantification of the frequency of live lymphocytes that are at the pro-B (B220^loCD43^loIgM⁻), pre-B (B220^loCD43⁻IgM⁻), or immature B (B220^loCD43⁻IgM⁺) stages in bone marrow of WT or Nemo^SA mice. The frequency of immature B cells that are Igκ⁺ or Igλ⁺ and the total numbers of cells of each population in the bone marrow also are quantified. (C) Representative flow plots of live, CD4⁻CD8⁻, lineage⁻ thymocytes expressing CD44 and/or CD25. (D) Quantification of lineage⁻ DN thymocytes at the DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), or DN4 (CD44⁻CD25⁻) stage in WT and Nemo^SA mice. (E) Representative flow plots of live thymocytes expressing CD4 and/or CD8 (top) and TCRβ or TCRδ (bottom) in WT or Nemo^SA mice. (F) Quantification of the frequency of live thymocytes that are DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺), CD4 (CD4⁺CD8⁻), CD8 (CD4⁻CD8⁺), αβ Τ cells (TCRβ⁺), or γδ T cells (TCRδ⁺) or the total number of αβ or γδ T thymocytes in WT or Nemo^SA mice. Individual points represent replicates (WT, n=6; Nemo^SA, n=6) from two independent experiments, and bars represent the mean ± SEM. Statistical significance was evaluated using multiple unpaired t-tests and the Holm-Šídák test for multiple comparisons. All comparisons were non-significant (corrected p-value > 0.05).

Finally, we assessed the potential consequences that expression of only endogenous Nemo^SA protein exerts upon Nemo-dependent gene expression changes induced in response to genotoxic DSBs. For this purpose, we isolated total bone marrow cells from individual Nemo^SA or WT mice and cultured them in the presence of etoposide or only vehicle (DMSO) for one or four hours. We then harvested cells, isolated mRNA, and used qRT-PCR to quantify levels of transcripts from specific genes. To validate that Nemo was expressed similarly in total bone marrow of WT and Nemo^SA mice, we measured Nemo mRNA and protein in the untreated sample by qRT-PCR and western blot. We found that Nemo mRNA was slightly reduced in Nemo^SA cells compared to WT cells (Fig. 2 A), yet Nemo protein expression was similar between genotypes (Fig. 2 B). To validate that etoposide treatment caused DSBs, we measured transcripts of the p21 cell cycle inhibitor gene that is upregulated in response to DSBs via ATM-dependent activation of the p53 transcription factor (5). At each timepoint, we detected equivalent levels of p21 transcripts in WT or Nemo^SA cells (Fig. 2 C). These data confirm that etoposide treatment induced DSBs and demonstrate that expression of only Nemo^SA protein does not interfere with ATM signaling to activate p53 in response to DSBs. As Nemo inactivation impairs the ability of etoposide or RAG DSBs to signal ATM-dependent repression of Rag1 and Rag2 transcripts in pre-B cells (23, 24), we next measured expression of these genes. At each timepoint, we detected similar decreased levels of Rag1 and Rag2 transcripts in WT or Nemo^SA cells, although Rag1 repression was slightly impaired after four hours of etoposide treatment in the Nemo^SA cells (Fig. 2 C). This result was unexpected as experiments in a mouse pre-B cell line indicated that ATM-dependent phosphorylation of human NEMO at serine 85 is necessary for etoposide-induced DSBs to activate NF-κB factors (12). However, it is possible that DSBs signal Nemo-dependent repression of Rag1 and Rag2 expression via factors other than NF-κB proteins. Accordingly, we also measured transcripts from the well-established NF-κB target gene Nfkbia, which encodes the IκBα protein that negatively controls NF-κB activity (43). Notably, in a Nemo-deficient mouse pre-B cell line, increased expression of Nfkbia induced by etoposide DBSs was only observed with wildtype NEMO expression and not with NEMO^S85A expression, indicating DSB-induced Nfkbia upregulation requires NEMO serine 85 (12). At each timepoint in our experiment, we observed the same levels of Nfkbia transcripts in response to etoposide DSBs in WT or Nemo^SA cells (Fig. 2 C), indicating that DSB-signaled activation of NF-κB-mediated gene transcription is intact in bone marrow cells that only express Nemo^SA protein. Together, these data demonstrate that expression of only endogenous Nemo^SA protein in primary mouse bone marrow cells supports Nemo-dependent, NF-κB-regulated gene expression changes following induction of DSBs. This finding was unexpected as we anticipated at least dampened induction of NF-κB target genes, including Nfkbia, in Nemo^SA cells given data from a mouse pre-B cell line showing that the same phosphomutant NEMO protein abrogates Nemo-dependent NF-κB activation in response to etoposide-induced DSBs. Accordingly, for reasons outlined below, we considered it possible that ATM-mediated phosphorylation of the remaining S/T-Q motifs within Nemo^SA protein could be sufficient to activate NF-κB transcription factors in response to genotoxic DSBs.

FIGURE 2.

Serine 85 of Nemo is dispensable for etoposide-induced DSBs to signal downregulation of Rag1 and Rag2 transcripts in total bone marrow. (A) qRT-PCR quantification of Nemo mRNA in total bone marrow from individual WT and Nemo^SA mice. The value for the transcript was calculated relative to Cd19. Individual points indicate replicates (WT, n=9; Nemo^SA, n=9) from three independent experiments and bars represent the mean ± SEM. (B) Western Blot data for Nemo protein in total bone marrow from WT and Nemo^SA mice. LaminA/C was used as a loading control. Each lane represents an individual replicate (WT, n=5; Nemo^SA, n=5). (C) qRT-PCR quantification of p21, Rag1, Rag2, and Nfkbia mRNA in total bone marrow from WT or Nemo^SA mice. Total bone marrow cells from individual mice were treated with etoposide (10 μg/mL in DMSO) or only DMSO and collected 1 or 4 hours after treatment. The value for each transcript was calculated relative to Cd19 and normalized to the baseline sample for each individual mouse. The data are presented as fold change ratios of relative levels of each transcript in etoposide treated cells compared with DMSO treated cells at 1 or 4 hours after treatment, making this value 1, which is represented by the dotted line in each graph. Individual points indicate replicates (WT, n=9; Nemo^SA, n=9) from three independent experiments and bars represent the mean ± SEM. Statistical significance was evaluated using multiple unpaired t-tests and the Holm-Šídák test for multiple comparisons. *, p < 0.05.

The serine residues of all Nemo SQ motifs are dispensable for normal B and T cell development and DSB-induced repression of Rag1 and Rag2 and upregulation of Nfkbia in B lineage cells.

Although NEMO^S85A expression in a mouse pre-B cell line abrogated NEMO-dependent NF-κB activation in response to etoposide but not LPS, individual serine to alanine mutation of three additional NEMO SQ motifs decreased NEMO-dependent NF-κB activation after treatment with either stimulus (12). Notably, one of these other three human NEMO SQ motifs, S156/Q157, is conserved in mouse Nemo protein (Fig. S1 A, B). The two other human NEMO SQ motifs are not conserved in mouse Nemo protein; however, mouse Nemo protein contains a third SQ motif, S116/Q117, in addition to the S85/Q86 and S156/Q157 motifs (Fig. S1 A, B). Mouse Nemo does not harbor any TQ motifs. We considered that ATM-dependent phosphorylation of any of the mouse Nemo SQ motifs could support Nemo-dependent, NF-κB-mediated changes in gene expression in response to DSBs. To address this possibility, we bred WT C57BL/6 male mice with Nemo^SA/SA female mice to obtain zygotes as hosts for CRISPR/Cas9 genomic editing to generate mice carrying a mutated Nemo gene that encodes Nemo protein with serine to alanine mutation at all three SQ motifs (S85A, S116A, and S156A). We identified one founder mouse with evidence of all three Nemo mutations. As we bred this founder with wild-type (WT) C57BL/6 mice, our genotyping of male progeny revealed that the three mutations were linked on the same X chromosome. We sequenced relevant regions of this allele and found the modified gene indeed encoded for Nemo protein carrying the S85A, S116A, and S156A mutations (Fig. S1 D, E). However, we also found a deletion of three nucleotides that results in loss of Nemo threonine 164 (T164) (Fig. S1 D, E). Accordingly, we refer to this as the Nemo^3SAΔT phosphomutant. We bred this Nemo^3SAΔT allele with C57BL/6 mice for an additional generation before breeding together males and females harboring the mutated X-linked Nemo^3SAΔT allele to generate mice that only express the Nemo^3SAΔT protein from one (male) or two (female) Nemo^3SAΔT alleles, hereafter referred to as Nemo^3SAΔT mice regardless of sex. We found that Nemo^3SAΔT mice were viable, fertile, and displayed no visual phenotypes (data not shown), indicating that the Nemo^3SAΔT protein does not have the severe consequences like the complete absence of Nemo protein.

We then investigated the potential effects that expression of only endogenous Nemo^3SAΔT protein has on the development of B and T cells. We detected no differences in the frequencies or numbers of pro-B, pre-B, or immature B cell populations, including immature Igκ⁺ and Igλ⁺ B cells, in the bone marrow of Nemo^3SAΔT compared to WT mice (Fig. 3 A, B). The frequency and total number mature splenic IgM⁺, Igκ⁺, or Igλ⁺ B cell populations were similar among Nemo^3SAΔT and WT mice (Fig. S2 D, E). We observed no differences in frequencies or numbers of cells at each thymocyte developmental stage (CD4⁻CD8⁻ double-negative (DN, DP, CD4⁺ SP, or CD8⁺ SP) or mature αβ or γδ T cells among Nemo^3SAΔT and WT mice (Fig. 3 C–F). In addition, Nemo^3SAΔT and WT mice have similar frequencies and numbers of mature splenic αβ and γδ Τ cells (Fig. S2 E, F). Together, these data demonstrate that expression of only endogenous Nemo^3SAΔΤ protein confers no obvious effects on the overall development or accumulation of mature B and T cells. Thus, the integrities of Nemo serines 85, 116, and 156 and threonine 164 are dispensable for the differentiation and maintenance of B and T lineage lymphocytes.

FIGURE 3.

Nemo serine 85, 116, 156, and threonine 164 are dispensable for normal overall B and T cell development. (A) Representative flow plots analyzing the frequency of cells at indicated B cell developmental stages in the bone marrow of WT or Nemo^3SAΔT mice. The bold gates indicate the population shown in the next panel. The first panel presents live lymphocytes that express B220 or CD43. The second panel shows B220^loCD43⁻ cells that express IgM. The third panel shows the IgM⁺ cells that express Igκ or Igλ. The frequencies are shown in the flow plots if those are the values quantified. The frequencies are not shown if the values graphed incorporate prior gating frequencies. (B) Quantification of the frequency of live lymphocytes that are at the pro-B (B220^loCD43^loIgM⁻), pre-B (B220^loCD43⁻IgM⁻), or immature B (B220^loCD43⁻IgM⁺) stages in bone marrow of WT or Nemo^3SAΔT mice. The frequency of immature B cells that are Igκ⁺ or Igλ⁺ and the total numbers of cells of each population in the bone marrow also are quantified. (C) Representative flow plots of live, CD4⁻CD8⁻, lineage⁻ thymocytes expressing CD44 and/or CD25. (D) Quantification of lineage⁻ DN thymocytes at the DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), or DN4 (CD44⁻CD25⁻) stage in WT or Nemo^3SAΔT mice. (E) Representative flow plots of live thymocytes expressing CD4 and/or CD8 (top) and TCRβ or TCRδ (bottom) in WT or Nemo^3SAΔT mice. (F) Quantification of the frequency of live thymocytes that are DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺), CD4 (CD4⁺CD8⁻), CD8 (CD4⁻CD8⁺), αβ Τ cells (TCRβ⁺), or γδ T cells (TCRδ⁺) or the total number of αβ or γδ T thymocytes in WT or Nemo^3SAΔT mice. Individual points represent replicates (WT, n=6; Nemo^3SAΔT, n=6) from two independent experiments, and bars represent the mean ± SEM. Statistical significance was evaluated using multiple unpaired t-tests and the Holm-Šídák test for multiple comparisons. All comparisons were non-significant (corrected p-value > 0.05).

We next determined the potential consequence that expression of only phosphomutant Nemo^3SAΔΤ protein asserts on Nemo-dependent gene expression changes triggered in response to genotoxic DSBs. For this purpose, we again quantified Nemo expression in primary bone marrow cells, as well as and p21, Rag1, Rag2, and Nfkbia transcripts in primary bone marrow cells treated with etoposide or vehicle alone for one or four hours. In untreated cells, Nemo mRNA and protein expression was similar in WT and Nemo^3SAΔT bone marrow cells (Fig. 4 A, B). At each timepoint, we detected equivalent levels of p21 transcripts between WT or Nemo^3SAΔT cells (Fig. 4 C), indicating that expression of only Nemo^3SAΔΤ protein does not impair ATM-mediated activation of the p53-p21 pathway. At each timepoint, we also detected similar decreased levels of Rag1 and Rag2 transcripts or increased levels of Nfkbia transcripts between WT and Nemo^3SAΔT cells (Fig. 4 C). These data demonstrate that expression of only endogenous Nemo^3SAΔΤ protein in primary mouse bone marrow cells supports Nemo-dependent, NF-κB-regulated gene expression changes in response to DSBs. As the Nemo^3SAΔT protein lacks all SQ motifs, our data indicate that potential ATM-dependent phosphorylation of Nemo SQ motifs is dispensable for Nemo-dependent activation of NF-κB-mediated gene expression changes triggered by genotoxic DSBs in primary bone marrow cells.

FIGURE 4.

Nemo serine 85, 116, 156, and threonine 164 are dispensable for etoposide-induced DSBs to signal downregulation of Rag1 and Rag2 transcripts in total bone marrow. (A) qRT-PCR quantification of Nemo mRNA in total bone marrow from individual WT and Nemo^SA mice. The value for the transcript was calculated relative to Cd19. Individual points indicate replicates (WT, n=9; Nemo^SA, n=9) from three independent experiments and bars represent the mean ± SEM. (B) Western Blot data for Nemo protein in total bone marrow from WT and Nemo^SA mice. LaminA/C was used as a loading control. Each lane represents an individual replicate (WT, n=5; Nemo^SA, n=5) from one experiment. (C) qRT-PCR quantification of p21, Rag1, Rag2, and Nfkbia mRNA in total bone marrow from WT or Nemo^3SAΔT mice. Total bone marrow cells from individual mice were treated with etoposide (10 μg/mL in DMSO) or only DMSO and collected 1 or 4 hours after treatment. The value for each transcript was calculated relative to Cd19 and normalized to the baseline sample for each individual mouse. The data are presented as fold change ratios of relative levels of each transcript in etoposide treated cells compared with DMSO treated cells at 1 or 4 hours after treatment, making this value 1, which is represented by the dotted line in each graph. Individual points indicate replicates (WT, n=3; Nemo^3SAΔT, n=3) from one independent experiment and bars represent the mean ± SEM. Statistical significance was evaluated using multiple unpaired t-tests and the Holm-Šídák test for multiple comparisons. All comparisons were non-significant (corrected p-value > 0.05).

The serine residues of all Nemo SQ motifs are dispensable for DSB-induced, NF-κB-dependent gene expression changes.

Our data indicate that Nemo SQ motifs are dispensable for changes in expression of DSB-induced, NF-κB-regulated genes indicates that other ATM/Nemo-dependent or -independent mechanisms signal these responses in bone marrow cells expressing only phosphomutant Nemo protein. To address this issue, we employed the ex vivo primary pre-B culture system that identified ATM- and Nemo-dependent gene expression changes upon DSBs in developing B cells (22–24). The culture of total bone marrow cells with IL7 cytokine leads to proliferation, expansion, and survival of pre-B cells such that after several days approximately 95% of cells are B lineage cells with the majority being pro/pre-B cells (44). IL7 suppresses Rag1 and Rag2 transcription, Igk chromatin accessibility, and Igk recombination in pre-B cells (22, 45–48). Upon IL7 withdrawal, pre-B cells in the culture arrest at the G1 cell cycle phase, induce Rag1 and Rag2 transcription, and activate Igk loci, which allows Igk recombination and resulting differentiation of immature B cells (22, 44, 46, 47). Expression of the anti-apoptotic EμBCL2 transgene in this culture system increases cellular survival for days after IL7 withdrawal (44). Accordingly, for our analyses, we bred Nemo^SA and Nemo^3SAΔT mice with EμBCL2⁺ (B⁺:Nemo⁺) mice to generate and use EμBCL2⁺:Nemo^SA (B⁺:Nemo^SA) and EμBCL2⁺:Nemo^3SAΔΤ (Β⁺:Nemo^3SAΔT) mice for cultures. To address Nemo-dependency, we cultured cells from Mb1Cre⁺:EμBCL2⁺:Nemo^flox (B⁺:Nemo⁻) mice that lack Nemo protein specifically in B lineage cells because we previously utilized this genetic model to elucidate Nemo-dependent gene expression changes in response to RAG DSBs (23). The addition of a small molecule inhibitor of ATM (ATMi) to bone marrow cultures has the same effect as genetic inactivation of ATM (22). To determine whether B lineage cells expressing phosphomutant Nemo protein still rely on ATM-dependent signals to alter expression of NF-κB-regulated genes upon DSBs, we treated ex vivo B cell cultures from phosphomutant Nemo mice with the ATMi. Furthermore, to determine if B lineage cells expressing phosphomutant Nemo protein still rely on IKKβ-mediated activation of NF-κB factors for DSB-induced changes in NF-κB target gene expression, we treated cells with a small molecule inhibitor of the IKKβ kinase (IKKβi).

For our experiments, we cultured total bone marrow cells from B⁺:Nemo⁺, B⁺:Nemo^SA, Β⁺:Nemo^3SAΔT, and B⁺:Nemo⁻ mice in the presence of IL7 over four days to enrich for pre-B cells. After four days, we removed IL7 for 48 hours to arrest cells at the G1 cell cycle phase and allow maximal expression of Rag1 and Rag2. We then split the cultures of each cell type into three aliquots and treated aliquots with ATMi, IKKβi, or DMSO vehicle control for two hours before treatment with etoposide or additional DMSO vehicle control. We harvested cells four hours after etoposide treatment, isolated mRNA, and used qRT-PCR to measure expression of genes known to be modulated by DSBs in pre-B cells through ATM- and Nemo-dependent mechanisms and/or regulated by NF-κB factors in response to other stimuli. To validate induction of DSBs and inactivation of ATM, we measured transcripts of the p21 gene that is upregulated by ATM-dependent activation of the p53 transcription factor following DSBs (5). Indeed, we observed that etoposide caused similar induction of p21 transcripts in B⁺:Nemo⁺, B⁺:Nemo^SA, Β⁺:Nemo^3SAΔT, and B⁺:Nemo⁻ cells, and that ATMi reduced this response in all three cell types (Fig. 5 A). Unexpectedly, we found that IKKβi also mildly reduced the ability of etoposide to induce p21 in all cell types (Fig. 5 A). As B⁺:Nemo⁻ cells exhibit normal p21 induction in response to etoposide alone, this finding reflects off-target effects of the IKKβi, that IKKβ modulates p21 expression independent of Nemo, or both. We observed that etoposide decreased Rag1 and Rag2 transcripts similarly in B⁺:Nemo⁺, B⁺:Nemo^SA, and Β⁺:Nemo^3SAΔT cells, but not at all in B⁺:Nemo⁻ cells (Fig. 5 A). This result indicates that DSB-induced repression of Rag1 and Rag2 transcripts is dependent on Nemo but not Nemo SQ motifs. The addition of ATMi or IKKβi antagonized the ability of etoposide to reduce Rag1 and Rag2 transcript levels in B⁺:Nemo⁺, B⁺:Nemo^SA, and Β⁺:Nemo^3SAΔT cells (Fig. 5 A), showing that DSB-induced repression of Rag1 and Rag2 expression is dependent on ATM and IKKβ signaling that does not require Nemo SQ motifs. Notably, addition of ATMi or IKKβi to etoposide treated B⁺:Nemo⁻ cells increased Rag1 and Rag2 transcript levels (Fig. 5 A), implying that other ATM- and IKKβ-dependent signals control Rag1 and Rag2 transcription as outlined in our Discussion. Nevertheless, these data demonstrate that DSB-induced repression of Rag1 and Rag2 expression depends on ATM, Nemo, and IKKβ, but not ATM-dependent phosphorylation of Nemo SQ motifs.

FIGURE 5.

Nemo serine 85, 116, 156, and threonine 164 are dispensable for Νemo- and ATM- dependent gene expression changes induced by DSBs in primary pre-B cell cultures. (A-B) qRT-PCR quantification of p21, Rag1, Rag2, Cd40, Cd69, Nfkbia, Nfkbie, Nfkb2, or RelB transcripts in primary pre-B cell cultures from B⁺:Nemo⁺, B⁺:Nemo^SA, B⁺:Nemo^3SAΔT, and B⁺:Nemo⁻ mice. Total bone marrow was cultured in the presence of IL7 for 4 days, then for 2 days without IL7. Remaining cells, enriched for pre-B/Immature B cells, were treated with DMSO, ATMi (15 μM KU55933 in DMSO), or IKKβi (10 nM TPCA-1 in DMSO) for 2 hours as a pretreatment and then cells were treated with either additional DMSO, (A-B) etoposide (10 μg/mL in DMSO), or (C) lipopolysaccharide (LPS; 15μg/mL in media) and collected 4 hours later. Individual points represent replicates (B⁺:Nemo⁺, n=4; B⁺:Nemo^SA, n=3; B⁺:Nemo^3SAΔT, n=3; B⁺:Nemo⁻, n=3) from three independent experiments that each included a B⁺:Nemo⁺ culture to normalize data. Bars represent the mean ± SEM. Each transcript was first normalized to Cd19 to account for cDNA input and then normalized to the DMSO only B⁺:Nemo⁺ cells in each individual experiment, making this value 1, which is represented by the dotted line in each graph. Statistical significance determined by Two-way ANOVA and Šídák’s multiple comparisons test comparing each value to the B⁺:Nemo⁺ etoposide treated sample (*, <0.0332; **, <0.0021; ***, <0.0002; ****, <0.0001).

To expand our work beyond Rag1 and Rag2 regulation, we assayed several other genes (Cd40, CD69, Nfkbia, Nfkbie, RelB, and Nfkb2) that have been shown to be induced by DSBs via ATM- and NF-κB-dependent mechanisms in primary pre-B cells (21, 49). Nfkbia and Nfkbie, which encode for the IκBα and IκBε proteins, respectively, are direct targets of canonical NF-κB factors downstream of Nemo activation and negatively regulate NF-κB activity (43). RelB and Nfkb2 encode the non-canonical NF-κB factors RelB and p100, respectively, with p100 the precursor of the active p52 factor (43). Although RelB and Nfkb2 are non-canonical NF-κB factors that are traditionally thought to be activated independent of Nemo, prior work in primary pre-B cells showed that the DSB-induced increase in expression of each of these genes relies on IκBα degradation and RAG DSB-induced expression of Nfkb2 requires Nemo (21, 23). These data suggest that DSB-induced expression of Nfkb2 and RelB may involve canonical NF-κB factor activation in primary pre-B cells. To determine whether the induction of these genes in response to DSBs requires ATM-dependent phosphorylation of Nemo SQ motifs, we assayed their transcript levels in our B⁺:Nemo⁺, B⁺:Nemo^SA, Β⁺:Nemo^3SAΔT, and B⁺:Nemo⁻ cells treated with etoposide and ATMi or IKKβi. We observed that etoposide induced Cd40, Cd69, Nfkbia, Nfkbie, Nfkb2, and RelB transcripts in B⁺:Nemo⁺, B⁺:Nemo^SA, and Β⁺:Nemo^3SAΔT cells, and that ATMi or IKKβi reduced this response in all three cell types (Fig. 5 B). However, etoposide had no effect on levels of Cd40, Cd69, Nfkbia, Nfkbie, Nfkb2, and RelB transcripts in B⁺:Nemo⁻ cells regardless of whether the cells were treated with ATMi or IKKβi (Fig. 5 B). These data demonstrate that the ability of DSBs to induce expression of these six NF-κB-regulated genes in B lineage cells depends on ATM and IKKβ activity yet does not require ATM-dependent phosphorylation of Nemo SQ motifs.

Phosphorylation of Nemo SQ motifs is dispensable for LPS induction of NF-κB target genes.

The study that demonstrated NEMO^S85A abrogates DSB-induced activation of NF-κB in a mouse pre-B cell line also showed that this mutation has no effect on NF-κB activation in response to LPS (12). As we found that Nemo^SA expression in primary mouse B lineage cells has no effect on Nemo-dependent regulation of NF-κB target genes in response to DSBs, we thought that it was important to determine the consequences of this Nemo mutation on LPS signaled gene expression changes. Moreover, while neither Nemo serine 116, serine 156, nor threonine 164 has been implicated in activation of NF-κB factors by pro-inflammatory signals, we thought it was valuable to evaluate the roles of these amino acids in LPS signaling. To address these issues, we used the ex vivo primary pre-B cell culture system. When we cultured cells from B⁺:Nemo⁺, B⁺:Nemo^SA, Β⁺:Nemo^3SAΔT, and B⁺:Nemo⁻ mice with IL7 for four days and removed IL7 for 48 hours, we also set up aliquots to be treated with LPS or vehicle. We harvested cells four hours after LPS treatment, isolated mRNA, and employed qRT-PCR to measure expression of the well-documented NF-κB target genes Nfkbia, Nfkbie, Nfkb2, and RelB (43). We observed that LPS increased the expression of these genes similarly in B⁺:Nemo⁺, B⁺:Nemo^SA, and Β⁺:Nemo^3SAΔT cells, but not had no effect on their expression in B⁺:Nemo⁻ cells (Fig. 5 C). These data demonstrate that neither the integrity nor the modification of the serines of the three Nemo SQ motifs or threonine 164 are required for LPS-signaled activation of NF-κB-mediated changes in gene expression.

Phosphorylation of Nemo SQ motifs is dispensable for Nemo-dependent DSB-induced gene expression changes in CD19⁺ splenic B cells.

As our data up to this point has not indicated a dominant role for ATM-mediated phosphorylation of Nemo in regulating DSB-induced or LPS-induced gene expression changes in primary pre-B cells, we decided to investigate a role for ATM-mediated phosphorylation of Nemo in primary CD19⁺ splenic B cells. To do this, we isolated CD19⁺ splenocytes from WT, Nemo^SA, Nemo^3SAΔT, and Nemo⁻ mice and cultured them for four hours with vehicle, LPS, or etoposide and measured known NF-κB target genes, Nfkbia, Nfkbie, Nfkb2, and RelB by qRT-PCR. We found that LPS increased expression of Nfkbia, Nfkbie, and Nfkb2 in WT, Nemo^SA, and Nemo^3SAΔT, whereas these genes did not increase in Nemo⁻ splenic B cells (Fig. 6 A), indicating that these LPS-induced, Nemo-dependent gene expression changes do not require ATM-mediated phosphorylation of Nemo in splenic B cells. Similarly, we found that etoposide treatment stimulated an increase in Nfkbia and Nfkbie expression in WT, Nemo^SA, and Nemo^3SAΔT splenic B cells that was impaired in Nemo⁻ splenic B cells (Fig. 6 B), consistent with induction of these DSB-responsive genes being Nemo dependent yet not relying on ATM-mediated phosphorylation of Nemo.

As these data only addressed four NF-κB target genes, we also utilized RNA sequencing to evaluate gene expression changes with an unbiased gene profiling approach. To do this, we isolated CD19⁺ splenocytes from WT, Nemo^3SAΔT, and Nemo⁻ mice, cultured them with vehicle (DMSO) or etoposide, and, after four hours of treatment, collected samples for RNA sequencing. Using these RNAseq data (File S1), we first identified 3,184 genes that were differentially expressed with etoposide treatment in WT CD19⁺ splenocytes, which we defined as having a log₂(Fold-Change Etoposide/DMSO) > 1 or < −1 and a p-value < 0.05 when comparing gene expression levels in etoposide and DMSO treated WT CD19⁺ splenocytes (Fig. 6 C, D). Next, we determined the subset of these genes that were Nemo-dependent, which included 1,005 genes that we defined as having a log₂(Fold-Change Etoposide/DMSO) < 1 or > −1 in Nemo⁻ CD19⁺ splenocytes and a p-value < 0.05 when comparing the etoposide-induced fold-change in expression in WT and Nemo⁻ CD19⁺ splenocytes (Fig. 6 C, D). We introduced the qualification that these Nemo-dependent genes be expressed above the 20th quantile of average expression to ensure observed changes in fold-change values were not an artifact of low expression. To depict the expression patterns of the genes in each of these subsets across different genotypes, we plotted the log₂(Fold-Change Etoposide/DMSO) value of each gene in WT CD19⁺ splenocytes against Nemo⁻ CD19⁺ splenocytes (Fig. 6 C) or Nemo^3SAΔΤ CD19⁺ splenocytes (Fig. 6 D). When the gene fold-change values are compared in WT and Nemo⁻ cells, we observe that the Nemo-dependent genes cluster near the x-axis, which indicates a lack of an etoposide-induced fold-change in expression in the Nemo⁻ cells consistent with their DSB-induced fold-change in expression requiring the expression of Nemo (Fig. 6 C). Conversely, when the Nemo-dependent gene fold-change values are compared in WT and Nemo^3SAΔT cells, these genes show concordance in that they cluster along a line with a slope of 1, indicating that the etoposide-induced fold changes are similar in WT and Nemo^3SAΔT CD19⁺ splenocytes (Fig. 6 D). Furthermore, only two Nemo-dependent genes (Tspan33 and Hsd11b) are also Nemo^3SAΔT-dependent, meaning a p-value < 0.05 when comparing the etoposide-induced fold-change in expression in WT and Nemo^3SAΔΤ CD19⁺ splenocytes. Collectively, these data illustrate in an unbiased manner that ATM-mediated phosphorylation of Nemo is largely dispensable for DSB-induced gene expression changes that rely on Nemo expression in B cells.

Discussion

Our work demonstrates that ATM-dependent phosphorylation of the three Nemo SQ motifs is dispensable for DSBs or LPS to cause changes in expression of NF-κB-regulated genes within primary mouse B lineage cells, including pre-B cells. This result was unexpected based on a prior study that showed ATM-mediated phosphorylation of human NEMO at serine 85 of a conserved SQ motif is vital for DSB-induced activation of NF-κB factors and NF-κB-mediated upregulation of Nfkbia transripts in a mouse pre-B cell line (12). There are plausible explanations for the marked difference in results between these two studies. One possibility is that the immortalization/transformation process rendered the pre-B cell line dependent on ATM-dependent phosphorylation of Nemo SQ motifs to activate NF-κB transcription factors in response to DSBs. In this context, the genetic or epigenetic signatures of this pre-B cell line could have inactivated the elucidated pathway through which ATM activates NF-κB factors independent of phosphorylating Nemo (13). A second possibility is that the ectopic expression of human NEMO protein, and perhaps at higher levels than normal for mouse Nemo protein, influences the signaling pathways activated by DSBs. For example, human NEMO could have a greater affinity for ATM than mouse Nemo and the resulting stronger interaction could diminish the ability of ATM to translocate out of the nucleus and activate the cytoplasmic signaling cascade necessary for IKK and NF-κΒ activation. Such a scenario has precedence in analyzing ATM function in DSB responses. Studies of human ATM in cell lines showed that autophosphorylation of ATM on serine 1981 is necessary for complete activation of ATM and its downstream signaling pathways (50). However, inactivation of the conserved serine in endogenous mouse ATM demonstrated that autophosphorylation at this site is dispensable for ATM activation and signaling in vivo (51). A third possibility is that differences in protein structure enable ATM to phosphorylate human NEMO at serine 85 or allow such ATM-phosphorylated human NEMO protein to functionally interact with other factors necessary for activation of NF-κB, with neither scenario operating for mouse Nemo protein. The reliance of human but not mouse lymphocytes on the XLF NHEJ factor for repair of DSBs induced by the RAG endonuclease offers precedence for a marked difference in the cellular DSB response between species (52, 53). Regardless of the reason for the distinct phenotypes observed with expression of human NEMO^S85A in a mouse pre-B cell line versus of mouse Nemo^S85A in primary mouse cells, the discrepancy emphasizes the critical need to corroborate key outcomes from immortalized cell lines with primary cells expressing endogenous proteins.

Our experiments reveal that primary mouse B lineage cells expressing endogenous Nemo protein mutants that cannot be phosphorylated at SQ motifs still rely on ATM and IKKβ kinase activity to induce NF-κB target genes in response to DSBs. This finding indicates that primary mouse B lineage cells employ other ATM-dependent mechanisms to activate NF-κB factors, such as the described ATM-mediated regulation of the cytoplasmic signaling cascade that targets IKK to activate NF-κB (13). Alternatively, ATM could activate NF-κB factors by phosphorylating other serine residues of the mouse Nemo protein as original work describing that ATM preferentially phosphorylates SQ motifs found that ATM is capable of phosphorylating serines of SF, SI, SP, and RXXS motifs (54). The mouse Nemo protein contains an SF, SP, and RXXS motif with serines at positions 169, 380, and 178, respectively, all of which would be available for ATM phosphorylation in our phosphomutant Nemo mice. In mouse cells, ATM might phosphorylate Nemo at one or more of these serines either normally in addition to one or more of the SQ motifs or as a compensatory mechanism when the relevant SQ motifs cannot be targeted. Otherwise, the ATM- and Nemo-dependent activation of NF-κB target genes could be mediated by phosphorylation of Nemo at additional serine or threonine residues by ATM-activated kinases, such as Chk2 (55), Akt (56), or DNA-PKcs (57). Notably, DNA-PKcs was shown to phosphorylate human NEMO serine 43, which is not followed by glutamine, to induce NF-κB activation in immortalized human cell lines with supporting evidence of this pathway in ex vivo culture of primary human macrophages derived from peripheral blood mononuclear cells (58). As serine 43 and the flanking residues are conserved in the mouse Nemo protein, it is conceivable that ATM might promote DNA-PKcs-mediated phosphorylation of Nemo at serine 43, at least when Nemo SQ motifs are not present. However, phosphorylation of Nemo by DNA-PKcs could be a cell type specific DSB response that does not function in B lineage cells. Future reductive experiments including in vitro kinase assays with wild-type or phosphomutant Nemo proteins and ATM, ATM-activated kinases, or DNA-PKcs could provide insights that would need to be followed up with in vivo models to determine the relevance of any targeted Nemo serine residues.

One of our main goals for generating the Nemo^SA and Nemo^3SAΔΤ phosphomutant mice was to establish a model in which DSB-induced activation of NF-κB was blocked while leaving intact NF-κB activation by all other stimuli. This would provide us a powerful experimental system to determine physiological roles of RAG DSB-induced activation of NF-κB-mediated gene expression changes in regulating lymphocyte specific processes. This approach would overcome the obstacle of studying these genetic programs in cells that completely lack either ATM or Nemo. These previous systems are not ideal because any phenotypes identified in cells with ATM inactivation could also be attributed to a loss of ATM-mediated DSB repair and any phenotypes identified with Nemo inactivation could be confounded by the loss of Nemo-dependent control of NF-κB transcription factors downstream of lymphocyte developmental signals. We had specific interest in leveraging phosphomutant Nemo mice to more specifically block ATM- and Nemo-dependent RAG DSB-induced repression of Rag1 and Rag2 transcription. If successful, this would have allowed us a more specific system to study the role of RAG DSB-induced Rag1 and Rag2 repression and transient feedback inhibition of V(D)J recombination in enforcing AgR gene allelic exclusion. Although our unexpected results prevented us from this objective, our experiments did provide insight into additional aspects of regulation of Rag1 and Rag2 expression within B lineage cells. When analyzing B⁺:Nemo⁻ pre-B cells, we found that Rag1 and Rag2 expression did not decrease with etoposide treatment and increased with ATMi or IKKβi treatment (Fig. 5 A). Furthermore, we observed that ATMi or IKKβi treatment increased basal levels of Rag1 and Rag2 transcripts in pre-B cells regardless of Nemo protein expression (data not shown). These data reinforce prior conclusions that basal levels of DSBs generated from normal cellular processes signal via ATM to prevent maximal Rag1 and Rag2 expression (24). However, our data expand upon this model as we found that ATM-driven repression of basal Rag1 and Rag2 expression is Nemo-independent but does rely on IKKβ. Thus, we postulate that two pathways might be targeting the Rag1/Rag2 locus to tightly regulate its expression; one by modulating expression of factors that stimulate Rag1 and Rag2 transcription and another that actively represses Rag1 and Rag2 expression. Experiments in an immortalized/transformed mouse pre-B cell line elucidated that ATM targets the FOXO1 transcription factor for degradation to suppress FOXO1-driven Rag1 and Rag2 transcription in response to DSBs (59). Moreover, our prior analyses of primary mouse pre-B cells lacking ATM or Nemo formulated a model wherein ATM and Nemo activate NF-κB factors that physically bind the Rag1/Rag2 locus to alter the chromatin environment and thereby repress transcription in response to DSBs (23). Our new data presented here is consistent with both models in that there is evidence of both an ATM- and IKKβ-dependent signaling pathway that does not require Nemo to dampen Rag1 and Rag2 expression and active repression of Rag1 and Rag2 expression in response to DSBs via signals mediated by ATM, IKKβ, and Nemo. Accordingly, the maximal Rag1 and Rag2 expression observed in ATMi or IKKβi treated B⁺:Nemo⁻ pre-B cells could be explained by the loss of Nemo completely disrupting active repression of Rag1 and Rag2 in response to DSBs in combination with ATM or IKKβ inhibition disrupting basal dampening of Rag1 and Rag2 transcription. Such putative tight control of Rag1 and Rag2 expression by DSB response mechanisms might be important for both enforcing AgR allelic exclusion and preventing genomic instability during V(D)J recombination. Elucidating the DSB response mechanisms that regulate Rag1 and Rag2 expression and evaluating their physiological relevance will require additional studies in primary mouse lymphocytes and appropriate new genetically modified mouse strains.

Key Points.

1. Nemo SQ motifs are not required for overall normal B/T cell development in mice.

2. Nemo SQ motifs are dispensable for DSB-dependent gene expression changes.