Abstract
Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1,2,3. Although uracils arising in DNA are accurately repaired1,2,3,4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1,2,3. Here we performed a genome-wide CRISPR–Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A–UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a−/− B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a−/− B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.
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Data availability
The CRISPR–Cas9 screen high-throughput sequencing data are available at the Gene Expression Omnibus (GSE184145). Source data for Figs. 1–4 can be found in Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank A. Frey for help with gRNA library subcloning and the CRISPR–Cas9 screen; B. Kavli for the anti-UNG1/2 antibody; L. Brino for help with gRNA library subcloning and CRISPR–Cas9 screen analysis; B. Jost, C. Thibault-Carpentier and D. Plassard for gRNA high-throughput sequencing on the GenomEast platform of IGBMC; D. Dembele, S. Sarnataro and N. Molina for help with Python scripts and gRNA extraction and counting; B. Heller for cell culture; C. Ebel and M. Philipps for cell sorting in the FlowCytometry Facility of IGBMC; M. Li for the GL-7 antibody, M. Selloum, D. Ali-Hadji and I. Gonçalves Da Cruz for animal care; W. Yu for generating Xrcc4−/− CH12 cells; and C. Goujon for the HIV1-VPR cDNA. J.M. and M.T. were supported by the Ministère de l’Enseignement Supérieur et de la Recherche, France, and the Fondation ARC. M.R. was supported by La Ligue Contre le Cancer. This study was supported by grants from the Fondation Recherche Médicale (Equipe FRM EQU201903007818 to B.R.-S.-M.), Institut National du Cancer (INCa_13852 to L.D. and B.R.-S.-M.), Fondation ARC (ARCPJA32020060002061 to B.R.-S.-M.), the Ligue Nationale contre le Cancer (Equipe labellisée to L.D.) and by the grant ANR-10-LABX-0030-INRT, a French state fund managed by the Agence Nationale de la Recherche under the program Investissements d’Avenir labelled ANR-10-IDEX-0002-02.
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Contributions
B.R.-S.-M. conceived and designed the study. M.R. generated CH12Cas9 cells, subcloned the gRNA library and performed the CRISPR–Cas9 screen with I.R. Screen data were analysed by B.R.-S.-M. and M.R. All CH12 knockout cell lines were generated and characterized by M.R. and J.M. with the help of A.A. The majority of experiments in CH12 cells and in Fam72a−/− mice were performed by M.R. and J.M. Igh FISH experiments were performed and analysed by C.L. All constructs were cloned by M.R. and J.M., with help from V.H. V.H. performed the nuclear/chromatin fractionations and provided assistance for western blot analysis. M.R. and J.M. performed the SHM experiments in CH12 cells with the help of V.H. M.R. and J.M. performed SHM analysis in germinal centre B cells, with the help of A.A. O.M. generated the AID off-target high-throughput sequencing libraries, which were sequenced and analysed by M.T. and E.P. K.C. performed and analysed SHM in DT40 cells. A.-S.T.-C. performed mRNA sequencing and analysed the data. B.L. generated VPR lentiviral particles. F.J. performed immunohistochemistry on spleen sections. E.P., K.T., M.C., S.G.C. and E.S. oversaw experiments. M.R., J.M., E.S. and B.R.-S.-M. wrote the manuscript. M.R., J.M., C.L., E.S., L.D. and B.R.-S.-M. edited the manuscript. The overall research was directed by L.D. and B.R.-S.-M.
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Extended data figures and tables
Extended Data Fig. 1 Setting up the CRISPR/Cas9 genome-wide knock-out screen.
a, Generation and validation of Cas9-expressing (CH12Cas9) cells. Clones were verified by Western blot for Cas9 expression, by PCR for Cas9-induced deletion of the enhancer γ1 (γ1E) and by flow cytometry for CSR. Clone #11 was chosen for the screen. Blots and data of CSR assay are representative of 3 experiments. b, The U6-gRNA sequence was amplified by PCR from the mBrie library and subcloned into pMX-28 using BamHI and NotI restriction enzymes to generate the pMX-mBrie gRNA library. gRNA representation was analyzed by high-throughput sequencing. c, Purity of IgM+ and IgA+ sorted populations was verified by flow cytometry. d, Number of reads at the Aicda and Fam72a genes in wild-type splenic B cells before and after 48h in culture with LPS and IL-4, as determined by mRNA-Seq. For gel source data, see Supplementary Fig. 1
Extended Data Fig. 2 FAM72A interacts with UNG2 and Fam72a-/- CH12 cells have no defects in proliferation, AID expression or switch region transcription.
a, Western blot analysis for Flag, UNG (UNG1 and UNG2) and β-Actin in wildtype and Fam72a-/- CH12 cells transduced with a retrovirus expressing FAM72A-Flag or FAM72AW125A-Flag before (Input) or after immunoprecipitation with a Flag-specific antibody (Pulldown). b, Schematic representation of the murine Fam72a locus and location of the gRNAs used to generate Fam72a-/- clones using CRISPR/Cas9-HF1. c, RT-PCR analysis for Fam72a and Igβ in Fam72a+/+ and Fam72a-/- CH12 cells cultured or not for 3 days with TGFβ, anti-CD40 antibody and IL-4 (CIT). Data are representative of three experiments. d, Western blot analysis for AID and β-actin in wild-type (pCH12) and Fam72a-/- CH12 cells cultured or not for 3 days with TGFβ, anti-CD40 antibody and IL-4 (CIT). e, CFSE dye-dilution analysis by flow cytometry in Fam72a+/+ and two independent Fam72a-/- CH12 cell clones cultured for 3 days with TGFβ, IL-4 and anti-CD40 antibody. Data are representative of three experiments. f, RT-qPCR analysis for GLTμ and GLTα in Fam72a+/+ and Fam72a-/- CH12 cells cultured for 3 days with TGFβ, IL-4 and anti-CD40 antibody. Triplicates were normalized to the abundance of Igβ and are expressed relative to Fam72a+/+, set as 1. Statistical significance was determined by a two-tailed Student’s t-test. Data are presented as mean ± s.d. and are representative of 3 experiments. For gel source data, see Supplementary Fig. 1
Extended Data Fig. 3 Robust B cell development and switch region transcription in Fam72a-/- mice.
a, Schematic representation of the murine Fam72a locus and strategy for the generation of Fam72a-/- mouse model. b, c, Flow cytometry and cellular analysis of B cell populations in the bone marrow (b) or the spleen (c) from Fam72a+/+ and Fam72a-/- mice, using the indicated cell surface markers. The data are representative of 5 mice per genotype. Horizontal line: mean values. d, Real-time qPCR analysis for germline transcripts at donor (GLTμ) and acceptor switch regions (GLTγ3, GLTγ1, GLTγ2b, GLTγ2a and GLTα) in Fam72a+/+ and Fam72a-/- splenic B cells cultured for 96h as in c. Expression is normalized to Igβ and is presented relative to expression in Fam72a+/+ B cells, set as 1. Mean of three mice per genotype + SEM were calculated following the rules for error propagation while calculating a ratio. Statistical analysis was performed using two-tailed Student’s t-test
Extended Data Fig. 4 FAM72A specifically controls UNG2 protein levels and its accession to chromatin via a proteasome-dependent mechanism.
a, RT-PCR analysis for Fam72a and Igβ in Fam72a+/+ and Fam72a-/- CH12 cells transduced (or not) with a retrovirus expressing FAM72A, FAM72AW125R or FAM72AW125A. Data are representative of three experiments. b, Flow cytometry analysis of IgA expression in Fam72a+/+ and Fam72a-/- CH12 cells expressing (or not) an UNG inhibitor (Ugi) and cultured for 3 days with TGFβ, anti-CD40 antibody and IL-4. Representative plots are shown. The percentage of IgA-expressing cells is indicated. c, Schematic representation of the murine Ung locus and location of the gRNAs targeting Ung2 (exon 1a; blue), Ung1 (exon 1b; purple) or Ung1/2 (exon4; black) used to generate Ung1-/-, Ung2-/-, Ung1-/- Ung2-/-, Fam72a-/- Ung1-/- and Fam72a-/- Ung2-/- CH12 cell clones using CRISPR/Cas9-HF1. Note, the gRNAs used to generate Ung1/2-/- CH12 cells target the same exon, which was deleted in Ung1/2-deficient mice. d, Western blot analysis for UNG (UNG1 and UNG2) and β-actin in wild-type, Fam72a-/-, Ung1/2-/-, Ung1-/-, Ung2-/-, Fam72a-/- Ung1-/- and Fam72a-/- Ung2-/- CH12 cells. Blots are representative of 3 experiments. e, Flow cytometry analysis of IgA expression in additional independent wildtype, Ung1-/-, Ung2-/-, Fam72a-/- Ung1-/- and Fam72a-/- Ung2-/- CH12 cells cultured for 72 h with TGFβ, IL-4 and anti-CD40 antibody. f, Western blot analysis for UNG (UNG1 and UNG2), NBS1 and Histone H3 on nuclear and chromatin fractions prepared from CH12 cells (pCH12) and Fam72a-/- CH12 cells expressing FAM72A or FAM72AW125R. Representative blots of 2 experiments. g, RT-qPCR analysis for Ung1, Ung2 and Igβ in Fam72a+/+ and Fam72a-/- splenic B cells cultured for 4 days with LPS, IL-4 and anti-IgD-Dextran. Triplicates were normalized to the abundance of Igβ and are expressed relative to Fam72a+/+ B cells, set as 1. Data are presented as mean of five mice per genotype ± s.d. Statistical analysis was performed using two-tailed Student’s t-test. h, Western blot analysis for UNG (UNG1 and UNG2) and β-Actin in wild-type and Fam72a-/- CH12 cells cultured in the presence of cycloheximide (CHX) and MG132. i, Quantification of the protein level of UNG2, relative to time point zero from 3 independent experiments. Data are presented as mean ± s.d. Statistical analysis was performed using two-tailed Student’s t test. For gel source data, see Supplementary Fig. 1
Extended Data Fig. 5 UNG2 protein levels correlate with the efficiency of CSR.
a, Western blot analysis for UNG (UNG1 and UNG2) and β-Actin protein levels in wild-type CH12 cells and primary B cells transduced with a retrovirus expressing FAM72A, FAM72AW125A or UNG2 and cultured with TGFβ, IL-4 and anti-CD40 antibody or LPS + IL-4 + anti-IgD-Dextran, respectively. b, Western blot analysis for UNG (UNG1 and UNG2) and β-actin in Fam72a-/- CH12 cells transduced with a retrovirus expressing HIV1-VPR (pMX-VPR), FAM72A and/or FAM72AW125A. c, Flow cytometry analysis of IgA expression in Fam72a-/- CH12 cells transduced with a retrovirus expressing HIV1-VPR (pMX-VPR), FAM72A and/or FAM72AW125A. Representative plots of three experiments are shown and quantified on the right. P values were determined using two-tailed Student’s t-test; see Statistics and Reproducibility section in Methods. n.s: not significant; **<0.01; ***<0.001. Horizontal line: mean values. d, Flow cytometry analysis of IgA expression in wild-type , Fam72a-/- and Ung1/2-/- CH12 cells transduced (or not) with a retrovirus expressing UNG2Degron and TIR1 and cultured for 72 h with TGFβ, IL-4 and anti-CD40 antibody in the presence or absence of auxin (IAA). Quantification is shown on the right. P-values were determined using two-tailed Student’s t-test; see Statistics and Reproducibility section in Methods. *<0.05; **<0.005; ***<0.0005. Horizontal line: mean values. Data are from three experiments. e, Western blot analysis for UNG (UNG1 and UNG2) and β-Actin in wild-type, Fam72a-/-, and Ung1/2-/-, transduced (or not) with a retrovirus expressing UNG2Degron and Tir1 and cultured in the presence or absence of auxin (IAA). Blots a, b and e are representative of 3 independent experiments. For gel source data, see Supplementary Fig. 1
Extended Data Fig. 6 Somatic hypermutation at the JH4 intron (JH4i).
a, Flow cytometry analysis of germinal center B cells (B220+ Fas+ GL-7+) isolated from the Peyer’s patches of unimmunized Fam72a+/+ and Fam72a-/- mice. Plots are gated on B220+ cells. b, Distribution of mutations at the JH4 intron (JH4i) in Fam72a+/+ (top) and Fam72a-/- (bottom) sequences obtained from germinal center B cells (B220+ Fas+ GL-7+) isolated from the Peyer’s patches (PPs) of unimmunized Fam72a+/+ and Fam72a-/- mice. c, Mean frequencies (at JH4i) of transition and transversion mutations at G/C and A/T base pairs, per sequence for each individual mouse analyzed. Data come from 5 mice of each genotype. p-value was determined using two-tailed Student’s t-test. Horizontal line: mean values
Extended Data Fig. 7 Somatic hypermutation at AID off-targets and Igh FISH strategy.
a, Tables depicting the mutation profiles observed for Bcl6, Cd83 and Pim1 in germinal center B cells (B220+ Fas+ GL-7+) isolated from the Peyer’s patches (PPs) of unimmunized Fam72a+/+ and Fam72a-/- mice. The respective number of nucleotides sequenced for each gene is also indicated. Data are from five mice per genotype. b, Schematic representation of the Igh locus with positions of the BACs used for generation of DNA FISH probes. Lower panel: DNA-FISH on representative metaphases from day 2 stimulated Fam72a-/- cells complemented with FAM72A or FAM72AW125A using the Igh C BAC probe (red) combined with Igh V BAC probe (green) and chromosome 12 paint (white). Yellow arrowheads point to broken or translocated chromosome 12
Extended Data Fig. 8 Working model for SHM and CSR in the presence or absence of FAM72A.
Immunoglobulin genes are diversified through Somatic Hypermutation (SHM) and Class Switch Recombination (CSR). They are both initiated by the deamination of cytosines in DNA induced by Activation Induced Cytidine Deaminase (AID). The resulting uracils are processed, mainly by the nuclear isoform of Uracil DNA Glycosylase (UNG2), and by proteins of the Base Excision Repair (BER) and Mismatch Repair (MMR) pathways to introduce mutations or double-stranded DNA breaks (DSBS) during SHM (left panel) or CSR (right panel). FAM72A interacts with UNG2 to control its physiological level by triggering its degradation and to enforce error-prone DNA repair. Consequently, deficiency in Fam72a leads to the specific upregulation of UNG2 and its accumulation on chromatin. This would enhance uracil excision, resulting in a reduction in the efficiency of SHM and CSR and enforced error-free DNA repair. Therefore, FAM72A controls the balance between error-prone and error-free DNA repair during antibody diversification.
Supplementary information
Supplementary Information
This file contains Supplementary Figures 1 and 2.
Supplementary Table 1
CRISPR–Cas9 screen results.
Supplementary Table 2
Igh FISH data.
Supplementary Table 3
Primers and gRNAs.
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Rogier, M., Moritz, J., Robert, I. et al. Fam72a enforces error-prone DNA repair during antibody diversification. Nature 600, 329–333 (2021). https://doi.org/10.1038/s41586-021-04093-y
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DOI: https://doi.org/10.1038/s41586-021-04093-y
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