Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination
- Department of Microbiology and Molecular Genetics, University of California, Davis, United States
- Department of Molecular and Cellular Biology, University of California, Davis, United States
- Graduate Group in Biostatistics, University of California, Davis, United States
- Center for Immunology and Infectious Diseases, University of California, Davis, United States
Figures
Figure 1 with 2 supplements
RNA-DNA hybrids are increased in Setx-/-Rnaseh2bf/f B cells during class switch recombination (CSR) to IgG1.
(A) Senataxin (SETX) and RNase H2 contribute to RNA:DNA hybrid removal; SETX helicase activity unwinds the nucleotide strands retaining both the RNA and DNA components while RNase H2 cleaves RNA, …
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Figure 1—source data 1
Uncropped raw dot blot analysis of R loop formation.
Genomic DNA extracted from WT, Rnaseh2bf/f, Setx-/-, and Setx-/-Rnaseh2bf/f cells was digested with restriction enzyme cocktail and run on dot blot, RnaseH1-treated Setx-/-Rnaseh2bf/f as a negative control.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig1-data1-v1.zip
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Figure 1—source data 2
Numerical data used to generate graphs in Figure 1C–E.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
Rnaseh2b deletion efficiency by CD19-cre.
(A) Deletion of genomic DNA measured by PCR from splenic B cells. (B) Transcription at the Rnaseh2b locus measured by RT-qPCR across exon 6, downstream of the Cre-mediated deletion site. (C) DNA:RNA …
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Figure 1—figure supplement 1—source data 1
Uncropped image of PCR analysis of Cre-mediated Rnaseh2b deletion.
PCR test of Rnaseh2b deletion efficiency on genomic DNA extracted from WT, Setx-/-, Rnaseh2bf/f, and Setx-/- Rnaseh2bf/f splenic B lymphocytes. Left and right represent two times results.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Numerical data used to generate graphs in Figure 1—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig1-figsupp1-data2-v1.xlsx
Figure 1—figure supplement 2
DNA:RNA hybrid formation at the Actb locus.
(A) Representative UCSC genome browser screenshot of DRIP-Seq signal in WT, Setx-/-, Rnaseh2bf/f, and Setx-/-Rnaseh2bf/f cells spanning 10 kb of the Actb locus (n = 2 mice/genotype). Locations of …
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Figure 1—figure supplement 2—source data 1
Numerical data used to generate graph in Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 1 supplement
Class switch recombination (CSR) is not reduced in Setx-/-, Rnaseh2bf/f, or Setx-/-Rnaseh2bf/f B cells.
Percentage of cells undergoing CSR to IgG1 72 hr (A) and 96 hr (B) post-stimulation with LPS/IL-4/α-RP105. (C) Representative flow cytometry analyses of IgG1+ and B220 expression in response to …
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Figure 2—source data 1
Numerical data used to generate graphs in Figure 2A, B, D and E.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Percentage of cells undergoing class switch recombination (CSR) to IgG1 72 hr post-stimulation with LPS/IL-4 alone.
Horizontal lines in dot plots indicate mean, and error bars show standard deviation. Statistical significance versus WT was determined by one-way ANOVA; each dot represents an independent mouse.
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Figure 2—figure supplement 1—source data 1
Numerical data used to generate graphs in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 3 supplements
Increased IgH damage is observed in Setx-/-Rnaseh2bf/f B cells.
(A) Frequency of spontaneous DNA damage in WT, Setx-/-, Rnaseh2bf/f, and Setx-/-Rnaseh2bf/f cells. (B) Frequency of spontaneous DNA damage at IgH. (C) Representative images of the types of …
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Figure 3—source data 1
Numerical data used to generate graphs in Figure 3A and B.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
B cell development in the bone marrow is normal in cells lacking senataxin (Setx) and RNase H2B.
(A) Flow cytometric gating of different developmental stages of B lymphopoiesis in the bone marrow (BM) of WT, Setx-/-Rnaseh2bf/f, and Setx-/-Rnaseh2bf/f mice (n = 4). Pre-pro-B cells are defined as …
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Figure 3—figure supplement 1—source data 1
Numerical data used to generate graphs in Figure 3—figure supplement 1B–D.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Ribonucleotide monophosphate (rNMP) incorporation and camptothecin (CPT) sensitivity in cells lacking senataxin (SETX) and RNase H2.
(A) Representative image of alkaline gel of genomic DNA (n = 3 independent mice/genotype). (B) Densitometry trace of representative alkaline gel shown in (A) with ImageJ software. (C) Representative …
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Figure 3—figure supplement 2—source data 1
Uncropped alkaline gel image from WT, Rnaseh2bf/f, Setx-/-, and Setx-/-Rnaseh2bf/f cells.
Genomic DNA from WT, Rnaseh2bf/f, Setx-/-, and Setx-/-Rnaseh2bf/f B cells was treated with NaOH for 3 hr before running the alkaline gel.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
Uncropped native gel image of WT, Rnaseh2bf/f, Setx-/-, and Setx-/-Rnaseh2bf/f B cells.
Genomic DNA from WT, Rnaseh2bf/f, Setx-/-, and Setx-/-Rnaseh2bf/f B cells was treated with NaCl (left) or NaOH (right) for 3 hr before running the TAE native gel.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-figsupp2-data2-v1.zip
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Figure 3—figure supplement 2—source data 3
Numerical data used to generate graphs in Figure 3—figure supplement 2E and F.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-figsupp2-data3-v1.xlsx
Figure 3—figure supplement 3
B cell proliferation and cell cycle in response to stimulation.
(A) Flow cytometry analysis of cell proliferation with CFSE staining. CFSE-labeled primary B cells were stimulated with LPS/IL-4/α-RP105, and cell division was measured by CFSE dye dilution at 72 hr …
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Figure 3—figure supplement 3—source data 1
Numerical data used to generate graph in Figure 3—figure supplement 3C.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig3-figsupp3-data1-v1.xlsx
Figure 4 with 1 supplement
RNase H2 activity rescues DNA:RNA hybrid levels in stimulated B cells.
(A) Total cell lysates were extracted from Setx-/-Rnaseh2bf/f cells stimulated for 96 hr with LPS/IL-4/α-RP105. Cells were infected with empty vector or retrovirus, expressing FLAG-RNaseH2B, then …
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Figure 4—source data 1
Uncropped western blot for RNaseH2B-FLAG expression in B cells under retroviral infection.
Immunoprecipitation and immunoblotting test for RNaseH2B-FLAG protein expression under retroviral infection in Setx-/-Rnaseh2bf/f B cells.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig4-data1-v1.zip
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Figure 4—source data 2
Uncropped alkaline gel from retrovirally infected cells.
Uncropped image of alkaline gel from Setx-/-, Setx-/-Rnaseh2bf/f EV, and Setx-/-Rnaseh2bf/f+FLAG-RNaseH2B cells; left and right represent two times results.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig4-data2-v1.zip
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Figure 4—source data 3
Numerical data used to generate graphs in Figure 4D and E.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Independent repeats of FLAG-RNaseH2B chromatin immunoprecipitation (ChIP).
Separate graphs for the four independent repeats of the FLAG-RNaseH2B ChIP shown in in Figure 4E.
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Figure 4—figure supplement 1—source data 1
Numerical data used to generate graphs in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
Activation-induced cytidine deaminase (AID) activity is required for persistent IgH breaks in Setx-/-Rnaseh2bf/f B cells.
(A) AID protein levels in WT B cells 72 hr post-stimulation to indicated isotypes (IgG1, LPS/IL-4/α-RP105; IgG3 with LPS/α-RP105; and α-RP105 alone). Actin served as a loading control. (B) …
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Figure 5—source data 1
Uncropped Western blots of activation-induced cytidine deaminase (AID) and actin protein expression.
AID protein expression in WT B cells 72 hr post-stimulation with different reagents. Actin served as a loading control.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig5-data1-v1.zip
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Figure 5—source data 2
Numerical data used to generate graphs in Figure 5B–F.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Aicda expression, enrichment, and recruitment in cells lacking senataxin (SETX) and RNase H2.
(A) Activation-induced cytidine deaminase (AID) mRNA quantitation by RT-qPCR in resting cells, LPS/IL-4/α-RP105, LPS/α-RP105, and α-RP105-only stimulated cells. Error bars show the standard …
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Figure 5—figure supplement 1—source data 1
Activation-induced cytidine deaminase (AID) expression test.
Whole-cell lysis from WT, Setx-/-, Rnaseh2bf/f, Setx-/-Rnaseh2bf/f, and Aicda-/- cells 72 hr post-stimulation to IgG1 were run on SDS-PAGE. Western blots were probed with an anti-AID antibody and anti-actin as loading control. Aicda-/- cells were used as a negative control.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Numerical data used to generate graphs in Figure 5—figure supplement 1A, C–E.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig5-figsupp1-data2-v1.xlsx
Figure 6 with 1 supplement
Altered switch junctions in in Setx-/-, Rnaseh2bf/f, and Setx-/-Rnaseh2bf/f B cells.
(A) Linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) analysis showing the percentage of sequenced junction events harboring blunt joins, microhomology …
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Figure 6—source data 1
Numerical data used to generate graphs in Figure 6D and E.
- https://cdn.elifesciences.org/articles/78917/elife-78917-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Features of Sμ-Sγ1 junction sites.
(A) Average number of reads from two runs of linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) spanning IgM-IgG1 junctions from WT, Setx-/-, Rnaseh2bf/f, …
Figure 7
Model for senataxin (SETX) and RNase H2 in promoting efficient class switch recombination (CSR).
When B cells are stimulated to undergo class switching, PolII-mediated transcription opens duplex DNA at recombining S regions. R loops formed during transcription promote activation-induced …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Rnaseh2bf/f | PMID:2802351 | RRID:MGI:5911393 | Dr. Axel Roers (University of Technology Dresden) |
| Genetic reagent (M. musculus) | Setx-/- | PMID:23593030 | RRID:MGI:5697060 | Dr. Martin F Lavin (Queensland Institute of Medical Research) |
| Genetic reagent (M. musculus) | Cd19cre | PMID:9092650 | RRID:MGI:5614310 | Dr. Klaus Rajewsky (University of Cologne) |
| Genetic reagent (M. musculus) | Aicda-/- | PMID:11007474 | RRID:MGI:2156156 | |
| Recombinant DNA reagent | Migr1-RNaseH2B-FLAG | This paper | N-terminally FLAG tagged mouse Rnaseh2b | |
| Antibody | Anti-S9.6 (mouse monoclonal) | Chedin lab | DRIP (1:500) Dot blot (1:1000) | |
| Antibody | IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (goat anti-mouse polyclonal) | Thermo Fisher | Cat# A21057 | Dot blot (1:5000) |
| Antibody | APC CD19 (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 561738 | Flow cytometry (1:100) |
| Antibody | PE-Cy7-CD43 (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 562866 | Flow cytometry (1:20) |
| Antibody | PE -IgM (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 562033 | Flow cytometry (1:100) |
| Antibody | BV421-CD11b (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 562605 | Flow cytometry (1:100) |
| Antibody | PerCP- CD45R/B220 (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 553093 | Flow cytometry (1:100) |
| Antibody | Anti-AID (mouse monoclonal) | Thermo Fisher | Cat# 39-2500 | CHIP (1:200) WB (1:500) |
| Antibody | Anti-KU70/KU80 (mouse monoclonal) | Invitrogen | Cat# MA1-21818 | CHIP (1:100) |
| Antibody | Anti-Rad52 (rabbit polyclonal) | ABclonal | Cat# A3077 | CHIP (1:500) |
| Antibody | Anti-RNA polymerase II (mouse monoclonal) | Abcam | Cat# AB5408 | CHIP (1:500) |
| Antibody | IgG- Isotype Control (mouse polyclonal) | Abcam | Cat# AB37355 | CHIP (1:500) |
| Antibody | Anti-CD180 (rat anti-mouse monoclonal) | BD Pharmingen | Cat# 552128 | CSR (1:2000) |
| Peptide, recombinant protein | Recombinant murine IL-4 | PeproTech | Cat# 214-14 | CSR (1:2000) |
| Peptide, recombinant protein | Lipopolysaccharides | MilliporeSigma | Cat# L2630 | CSR (1:2000) |
| Commercial assay or kit | Beckman Coulter AMPURE XP | Beckman Coulter | Cat# A63881 | Size selection |
| Commercial assay or kit | SPHERO AccuCount Blank Particles | Spherotech | Cat# ACBP-50-10 | Flow cytometry |
Additional files
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Supplementary file 1
Summary of individual fluorescent in situ hybridization (FISH) results for experiments shown in Figures 3 and 5.
- https://cdn.elifesciences.org/articles/78917/elife-78917-supp1-v1.xlsx
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Supplementary file 2
Primers used in qPCR and junction analysis.
Source data files
- https://cdn.elifesciences.org/articles/78917/elife-78917-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/78917/elife-78917-mdarchecklist1-v1.pdf
