The structure-selective endonucleases GEN1 and MUS81 mediate complementary functions in safeguarding the genome of proliferating B lymphocytes
- Immunology Program, Memorial Sloan Kettering Cancer Center, United States
- Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, United States
- Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, United States
- Gerstner Sloan Kettering Graduate School of Biomedical Sciences, United States
- Biochemistry, Cellular and Molecular Biology Allied Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, United States
Figures
Figure 1 with 1 supplement
B cell development in the bone marrow (BM) and spleen of Mb1-Cre Gen1−/− Mus81fl/fl mice.
(A, B) mRNA expression of Gen1 (A) and Mus81 (B) in developing and mature B cell subsets in the BM, spleen, and peritoneal cavity. FO: follicular, MZ: marginal zone (GEO Accession: GSE72018). (C) Spleens harvested from 4-month-old mice of the indicated genotypes. (D, E) Gating strategy (D) and absolute quantification (E) of live splenocytes, splenic B220+CD43–B, and B220+CD43+B cells. (F) Gating strategy of total B (B220+TCRβ–), total T (B220–TCRβ+), mature recirculating, immature, pro-B (fractions B and C), early pre-B (fraction C′), and late pre-B cells (fraction D). (G–I) Absolute quantification of BM cellularity, total B, and total T cell populations (G), of mature recirculating and immature B cell populations (H), and of B cell populations belonging to fractions B to D (I). Data in (E) and (G–I) are from four independent experiments with 18–22 mice per genotype. Bars display the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values were enumerated using ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing wherein all means were compared to the Mb1-Cre Gen1−/− Mus81fl/fl group. TPM, transcripts per million.
Figure 1—figure supplement 1
Temporal expression of Gen1 and Mus81, genetic structure of Gen1−/− and Mus81fl/fl alleles, Mendelian frequencies of Gen1−/− and Mus81−/− offsprings, and genotype validation of Gen1−/− Mus81fl/fl and Mb1-Cre Gen1+/− Mus81fl/fl pro-B cells.
(A) Relative quantification of Gen1 and Mus81 mRNA transcript levels by RT-qPCR in wild-type mouse B cells stimulated in lipopolysaccharide (LPS)+interleukin-4 (IL-4) culture from 24- to 96-hr post-stimulation. The expression level of the transcripts was normalized to the 0-hr time point. (B) Schematic of gene-targeting in generating the constitutive Gen1−/− mouse model. The reading frame of exon 4 is disrupted with a floxed neomycin-resistance gene cassette that was subsequently excised upon Cre recombinase expression. (C) Tables depicting the observed and expected frequencies of the genotypes of pups generated from the indicated breeding schemes: G and M represent the wild-type alleles of Gen1 and Mus81, respectively, whereas g and m represent the null alleles of Gen1 and Mus81; d.f. denotes degree(s) of freedom. (D) Schematic of the Mus81fl/fl allele in which exons 3–10 are flanked by loxP sites. When Cre recombinase is expressed under a tissue-specific promoter, exons 3 through 10 are excised, generating a null allele. The FRT site is a remnant of a pair that previously flanked a lacZ reporter-neomycin selection cassette. (E) Copy number quantification relative to control (Gen1+/− Mus81fl/fl) of unrecombined Mus81fl/fl allele in fractions B+C cells isolated from the bone marrow of mice of the indicated genotypes. (F) Relative Gen1 and Mus81 mRNA expression in sorted fractions B+C cells. Data in (A) are from three independent experiments with seven mice per time point. Data in (E) and (F) are from two independent experiments with 4–6 mice per genotype.
Figure 2 with 2 supplements
Homeostatic and induced GC responses in Cd23-Cre Gen1−/− Mus81fl/fl mice.
(A–C) Homeostatic GC response in mesenteric lymph nodes and Peyer’s patches. (A) Flow cytometric plots depicting the GL7+Fas+GC B cells in the Peyer’s patches of mice for each indicated genotype. (B, C) Quantification of the frequencies and absolute numbers of the GL7+Fas+GC B cell population in the mesenteric lymph nodes (B) and Peyer’s patches (C). (D–F) Evaluation of GC response during SRBC challenge. (D) Representative plots of the GL7+Fas+GC B cell population isolated from the spleen of mice immunized with SRBC for each indicated genotype. (E, F) Quantification of the frequencies and absolute numbers of GC B cells (E) and of total B cells (F) in the spleen of SRBC-immunized mice. (G, H) Assessment of induced GC response upon NP-CGG challenge in the Cd23-Cre Gen1−/− Mus81fl/fl mice at day 21 post-immunization. (G) Quantification of the percentage and absolute count of GL7+Fas+GC B cells in the spleen. (H) Frequencies and absolute numbers of live B220+ B cells in the spleen of PBS-treated and NP-CGG-immunized mice. Data in (B) and (C) are from four independent experiments with 11–21 mice per genotype. Data in (E) and (F) are from three independent experiments with 4–9 mice per genotype for the PBS group and 7–20 mice per genotype for the SRBC group. Data in (G) and (H) are from three independent experiments with 5–13 mice per genotype in the PBS group and 10–25 mice in the NP-CGG group. Bars represent the arithmetic mean and the error bars depict the 95% confidence interval of the measured parameters. For (B) and (C), p values were computed by ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing in which the means were compared to the Cd23-Cre Gen1−/− Mus81fl/fl group. For (E–H), ordinary two-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing was used to calculate the p values. All means were compared within each treatment group to the Cd23-Cre Gen1−/− Mus81fl/fl cohort.
Figure 2—figure supplement 1
Steady-state phenotyping and quantification of the splenic and bone marrow (BM) B cell populations in the Cd23-Cre Gen1-/- Mus81fl/fl mice.
(A) Quantification of BM cellularity and percentage of total B and T cell populations in the BMs of mice of the indicated genotypes. (B) Gating strategy of the B-cell populations in the BM: mature recirculating, immature, late-pre-B, and pro-B/early pre-B cells. (C) Quantification of the percentage among live B220+ B cells and absolute counts of mature recirculating and immature B cells. (D) Quantification of frequencies and absolute counts of late pre-B and pro-B/early pre-B cells among live B220+ B cells. (E) Representative flow cytometric plot depicting gating strategy of B220+CD43–B, B220+CD43+B, and non-B (B220–CD43+) cells in the spleen. (F, G) Quantification of the absolute number of live splenocytes (F), and of the percentage of splenic CD43–B, CD43+B, and non-B cells (G) in the mice of the indicated genotypes. (H) Gating strategy of the various splenic B cell subpopulations: marginal zone (MZ), follicular (FO), transitional-1 (T1), and transitional-2 (T2) B cells. (I) Frequencies of follicular and marginal zone B cells among total splenic B220+ cells. (J) Frequencies of T1 and T2 B cells among whole B220+B cells in the spleen. (K) Relative expression of Gen1 and Mus81 mRNA transcripts in LPS+IL-4-activated B cells of the indicated genotypes at 48 hr after stimulation. Data in (A–J) are from three independent experiments with 11–18 mice per genotype. Data in (K) are from three independent experiments with six mice per genotype. Bars depict the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values were calculated with ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing. All means were compared to the Cd23-Cre Gen1−/− Mus81fl/fl group.
Figure 2—figure supplement 2
Assessment of Mus81 deletion efficiency in the germinal center by genomic qPCR and RT-qPCR analyses and examination of class switching in induced GCs in Cd23-Cre Gen1−/− Mus81fl/fl mice.
(A) Quantification of the copy number relative to control (Gen1+/− Mus81fl/fl) of unrecombined Mus81fl/fl allele in GC and non-GC B cells sorted from the Peyer’s patches. (B, C) Relative measurement of Mus81 and Gen1 mRNA transcript levels in GC and non-GC B cells purified from the Peyer’s patches. (D–G) Quantification of the frequency and absolute number of IgG1-switched GC B cells in the spleen of SRBC-immunized mice (D, E) and in the spleen of NP-CGG-challenged mice (F, G). Data in (A–C) are from two independent experiments with 6–7 mice per genotype. Data in (D) and (E) are from three independent experiments with 4–9 mice per genotype for the PBS group and 7–20 mice per genotype for the SRBC group. Data in (F) and (G) are from three independent experiment with 5–13 mice per genotype in the PBS group and 10–25 mice in the NP-CGG group. Bars display the arithmetic mean and the error bars depict the 95% confidence interval of the measured parameters. Ordinary two-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing was used to calculate the p values. All means were compared within each treatment group to the Cd23-Cre Gen1−/− Mus81fl/fl cohort.
Figure 3 with 2 supplements
Growth, proliferation, cell cycle, and cell death profiles of ex vivo-stimulated DKO B lymphocytes.
(A) Growth curve of LPS+IL-4 (LI)-activated B cell culture. (B) Representative CTV dilution profiles of ex vivo-activated B cells at 0- and 72-hr post-stimulation for all indicated genotypes and culture conditions. (C) Frequency of live B220+ cells in each division. (D) Absolute number of undivided (division zero) cells after 72 hr of LI culture. (E) Class switching of B lymphocytes to IgG1 at 72- and 96-hr post-activation quantified as absolute number of live IgG1-expressing B cells. (F) Representative flow cytometry plots delineating the cell cycle stages of the cultured B cells based on EdU positivity and nuclear DNA content as determined by the intensity of DAPI staining. (G) Frequency of live B cells in G0/G1, S, and G2/M phases after 48, 72, and 96 hours of activation. (H) Fraction of dead cells among B220+ singlets at 72 and 96 hr after LI activation. (I) Percentage of cleaved caspase-3+ cells among live cells at 48-, 72-, and 96-hr post-stimulation. (J) Frequency of cleaved caspase-3+ cells among live B220+ cells in G0/G1, S, and G2/M phases after 48, 72, and 96 hr of culture. Data in (A) are from four independent experiments with nine mice per genotype. Data in (C) and (D) are from four independent experiments with 7–9 mice per genotype. Data in (E) are from seven experiments with 17 mice per genotype. Data in (G) are from six independent experiments with 11–12 mice per genotype. Data in (H) are from seven experiments with 17 mice per genotype. Data in (I) are from five independent experiments with 9–10 mice per genotype. Data in (J) are from three independent experiments with six mice per genotype. Bars display the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values were determined using ordinary two-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing wherein the mean of the Cd23-Cre Gen1−/− Mus81fl/fl group was compared to the rest.
Figure 3—figure supplement 1
Ex vivo characterization of DKO cells following LPS and LPS+TGF-β+anti-IgD-dextran (LTD) stimulations.
(A) Growth curve of LPS- and LTD-activated B cells. (B) Percentage of live B220+ cells in each division. (C) Absolute number of undivided (division zero) cells after 72 hr in LPS or LTD culture. (D) Class switching of B lymphocytes to IgG3 or IgA at 72- and 96-hr post-activation. Class-switched cells were quantified as absolute numbers of live IgG3- or IgA-expressing cells. (E, F) Cell cycle analysis of ex vivo-activated B cells. Frequency of cells among live B220+B cells in G0/G1, S, and G2/M phases at 48, 72, and 96 hr of LPS (E) or LTD (F) culture. Data in (A) are from four independent experiments with nine mice per genotype. Data in (B) and (C) are from three independent experiments with six mice per genotype. Data in (D) are from four independent experiments with nine mice per genotype. Data in (E) and (F) are from six independent experiments with 12 mice per genotype. Bars display the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values were determined using ordinary two-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing wherein the mean of the Cd23-Cre Gen1−/− Mus81fl/fl group was compared to the rest.
Figure 3—figure supplement 2
Cell death profiles of DKO cells stimulated in LPS and LTD cultures.
(A) Frequency of dead cells quantified as percentage of B220+ singlets staining for the viability dye after 72 and 96 hr of culture in media with LPS or LPS+TGF-β+anti-IgD-dextran (LTD). (B) Apoptosis in ex vivo B cell cultures. Percentage of cleaved caspase-3+ cells within the live B cell population at 48-, 72-, and 96-hr post-stimulation. (C, D) Cell cycle-specific determination of apoptosis in ex vivo-activated B cells. Frequency of cleaved caspase-3+ cells among live B220+ cells in each phase at 48, 72, and 96 hr of LPS (C) or LTD (D) culture. Data in (A) are from four independent experiments with nine mice per genotype. Data in (B) are from five independent experiments with 8–10 mice per genotype. Data in (C) and (D) are from two (48 hr) or three (72- and 96-hr) independent experiments with 4–6 mice per genotype. Bars represent the arithmetic mean and the error bars depict the 95% confidence interval of the measured parameters. P values were calculated using ordinary two-way ANOVA analysis with Dunnett’s multiple comparisons test in which the means were compared to that of the Cd23-Cre Gen1−/− Mus81fl/fl cohort.
Figure 4 with 2 supplements
RNA-seq and gene set enrichment (GSEA) analyses of activated control and DKO B cells.
(A) Volcano plot depicting the differential gene expression between control and DKO cells. Labeled squares indicate the top 15 most significantly upregulated genes and the labeled triangles are the 10 most downregulated genes in DKO cells. Using the Hallmark Gene Sets as reference, genes labeled in blue are categorized as genes in the p53 pathway while those labeled in red are defined as interferon alpha (IFN-α) response genes. Purple symbols mark Gen1 and Mus81. (B) Graph depicting the list of Hallmark Gene Sets that are differentially expressed between control and DKO cells (FDR<0.05). Value in each bar denotes the FDR for that gene set. (C) Heatmaps displaying the relative expression of genes within the indicated Hallmark Gene Sets that meet the expression cutoff (log2 fold change>1 for p53 pathway; >0.5 for apoptosis and interferon alpha response; <–0.3 for G2/M checkpoint; with FDR<0.05). Data are from six mice per genotype. The labels ‘1’ and ‘2’ represent male and female mice, respectively, and ‘A’ to ‘C’ indicate the three experimental ex vivo groups.
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Figure 4—source code 1
R script to generate Figure 1A, Figure 4, and Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-code1-v3.zip
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Figure 4—source code 2
Bash script to clean and align FASTQ files.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-code2-v3.zip
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Figure 4—source code 3
Bash script to count gene alignments.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-code3-v3.zip
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Figure 4—source data 1
Differential expression between control and DKO B cells.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-data1-v3.csv
Figure 4—figure supplement 1
Transcriptomics and gene set enrichment (GSEA) analyses of ex vivo-activated control, GKO, and MKO B cells.
(A) Table listing the first five genes and the corresponding log2 fold change, p value, and FDR generated from the DESeq analysis comparing Gen1+/− Mus81fl/fl (control) and Gen1−/− Mus81fl/fl (GKO) B cells. (B) Volcano plot displaying the differential gene expression between control and Cd23-Cre Gen1+/− Mus81fl/fl (MKO) cultures at 48-hr post-stimulation. Purple dots indicate the top 10 significantly upregulated genes, and the green dots indicate genes that are de-enriched. (C) Table listing the genes that are differentially expressed between GKO and MKO B cells. (D) GSEA plots of the Hallmark Gene Sets enriched in DKO cells. (E) GSEA plots of the Hallmark Gene Sets de-enriched in DKO cells. (F) Heatmap depicting the genes within the indicated Hallmark Gene Sets that had a log2 fold change of ≤–0.3 and FDR of <0.05 in DKO cells relative to control. Data are from six mice per genotype. The labels ‘1’ and ‘2’ represent male and female mice, respectively, and ‘A’ to ‘C’ denote the three experimental ex vivo groups.
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Figure 4—figure supplement 1—source data 1
Differential expression between control and GKO B cells.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-figsupp1-data1-v3.csv
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Figure 4—figure supplement 1—source data 2
Differential expression between control and MKO B cells.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-figsupp1-data2-v3.csv
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Figure 4—figure supplement 1—source data 3
Differential expression between GKO and MKO B cells.
- https://cdn.elifesciences.org/articles/77073/elife-77073-fig4-figsupp1-data3-v3.csv
Figure 4—figure supplement 2
In vivo and ex vivo phenotyping of DKO Trp53-WT, DKO Trp53-Het, and DKO Trp53-KO B cells.
(A, B) Absolute number of total B cells and frequency of GC B cells in the mesenteric lymph nodes (A) and Peyer’s patches (B) of mice for each indicated genotypes. (C) Growth curve of LPS+IL-4 (LI)-activated splenic B cell culture. (D) Representative CTV dilution plots of activated B cells at 72-hr post-stimulation. (E) Frequency of IgG1, IgG3, and IgA class-switched cells after 72 and 96 hr of stimulation in LI, LPS, and LTD cultures, respectively. (F) Cell cycle profile of LI-stimulated B cells after 48, 72, and 96 hr of culture represented by the frequency of live B cells in G0/G1, S, and G2/M phases. (G) Frequency of dead B cells at 48-, 72-, and 96-hr post LI-activation. (H) Fraction of live B cells undergoing apoptosis as defined by positive staining for anti-cleaved caspase-3 antibody after 48, 72, and 96 hr of LI culture. (I) Cell cycle phase-specific assessment of apoptosis. Frequency of cleaved caspase-3+ B cells in G0/G1, S, and G2/M phases at 48, 72, and 96 hr post-stimulation in LI culture. Data in (A–I) are from two independent experiments with 5–10 mice per genotype. Bars represent arithmetic mean and error bars denote 95% confidence intervals of the measured values. P values were computed using one-way ANOVA analysis (A, B) and two-way ANOVA analysis (E–I) with Dunnett’s multiple comparison test wherein the mean of the DKO Trp53-KO group was compared to the others.
Figure 5 with 2 supplements
Metaphase chromosomal analysis of activated DKO B cells.
(A) Representative image of a DKO metaphase spread with arrows indicating chromosomal breaks, fragments, and fusions in metaphases of activated DKO B cells. (B) Quantification of the average number of chromosomal aberrations across 45–50 metaphase spreads prepared from each B cell culture. (C) Percentage breakdown of metaphases exhibiting 0 to greater than 5 chromosomal aberrations. (D) Fraction of metaphases containing the indicated types of chromosomal abnormalities. Total percentage per genotype exceeds 100% as some metaphases exhibit more than one type of abnormality. (E) Proportion of chromatid and chromosome breaks among the 163 breaks observed in DKO metaphase spreads exhibiting at least one break. (F) Tel-FISH images of DKO metaphases highlighting the proximal location of the chromosomal damage to the telomeres. Note that the events labeled as dicentrics here and in Figure 5—figure supplement 1 may include chromosomes with residual cohesins remaining at a repaired DSB and those with condensins that persist after loading onto the chromosomal arms during mitotic entry. Data in (B–E) are from three independent experiments with 5 mice (totaling between 215 and 235 metaphase spreads) per genotype. For (C), the percentages are the average of the data combined from all five mice. Bars display the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values were computed with ordinary one-way ANOVA analysis (B, D) and the Kruskal-Wallis test (C) with Dunnett’s multiple comparisons test without pairing. Means of all groups were compared to that of Cd23-Cre Gen1−/− Mus81fl/fl.
Figure 5—figure supplement 1
Telomere FISH analysis of metaphase spreads prepared from ex vivo-activated DKO B cells.
Representative immunofluorescence images depicting in the context of stained telomeric DNA the various aberrations occurring in DKO B cells activated for 48 hr with LPS+IL-4.
Figure 5—figure supplement 2
Proliferation dynamics, viability, and chromosomal analyses of DKO Aicda-Het and DKO Aicda-KO ex vivo cultures.
(A) Growth curves of activated DKO Aicda-Het and DKO Aicda-KO splenic B cells. (B, C) Proliferation dynamics of DKO Aicda-Het and DKO Aicda-KO B cells in ex vivo cultures. Representative CTV dilution plot at 72-hr post-stimulation (B) and percentage of cells in each cell division (C) quantified at 72 hr after activation with LPS+IL-4, LPS, and LTD. (D) Frequency of cleaved caspase-3+ cells among ex vivo-activated B cells at 48-, 72-, and 96-hr post-stimulation. (E) Quantification of the average number of chromosomal aberrations present across all metaphase spreads of B cells isolated from each mouse of the indicated genotypes and activated with LPS+IL-4 for 48 hr. Data in (A–C) are from five independent experiments with 14 mice per genotype. Data in (D) are from three independent experiments with 9–11 mice per genotype. Data in (E) are from three independent experiments with six mice for each indicated genotype. Between 298 and 300 metaphase spreads were examined for each genotype. Bars display the arithmetic mean and error bars represent the 95% confidence interval of the measured parameters. P values in (C) and (D) were calculated using mixed-effects two-way ANOVA analysis with Šidák multiple comparisons test. For (E), p values were enumerated using ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test without pairing.
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The structure-selective endonucleases GEN1 and MUS81 mediate complementary functions in safeguarding the genome of proliferating B lymphocytes
eLife 11:e77073.
https://doi.org/10.7554/eLife.77073
