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. 2022 Dec 21;11:e78917. doi: 10.7554/eLife.78917

Figure 1. 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, retaining only the DNA strand. (B) Dot blot analysis of R loop formation: twofold serial dilutions of genomic DNA starting at 1 µg were arrayed on a nitrocellulose membrane and probed using the S9.6 antibody; RNase H treatment of the Setx-/-Rnaseh2bf/f sample was used as the negative control. (C) Quantitation of 0.5 µg, 0.25 µg, and 0.125 µg dot blot from (B) using ImageJ; values were normalized to WT, set as 1. Figures are expressed as fold change relative to WT. Error bars are the standard deviation between different experiments; *p<0.05 comparing different genotypes using multiple t-test (n = 3 mice/genotype). (D) Diagram of the IgH locus showing the location of PCR products used for chromatin immunoprecipitation (ChIP) and DNA:RNA hybrid immunoprecipitation (DRIP) assays. DRIP assay was performed with S9.6 antibody using primary B cells after 72 hr of stimulation to IgG1 with LPS/IL-4/α-RP105; RNase H treatment of Setx-/-Rnaseh2bf/f sample was used as the negative control. Relative enrichment was calculated as ChIP/input, and the results were replicated in three independent experiments. Error bars show standard deviation; statistical analysis was performed using one-way ANOVA (n = 3 mice/genotype). (E) Diagram of the IgH locus showing the location of PCR products for germline transcription under IgG1 stimulation with LPS/IL-4/α-RP105. Real-time RT-PCR analysis for germline transcripts (Ix-Cx) at donor and acceptor switch regions in WT, Setx-/-, Rnaseh2bf/f, and Setx-/- Rnaseh2bf/f splenic B lymphocytes cultured for 48 hr with LPS/IL-4/α-RP105 stimulation. Expression is normalized to CD79b and is presented as relative to expression in WT cells, set as 1. Error bars show standard deviation; statistical analysis was performed using multiple t-test (n = 4 mice/genotype).

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.
Figure 1—source data 2. Numerical data used to generate graphs in Figure 1C–E.

Figure 1.

Figure 1—figure supplement 1. Rnaseh2b deletion efficiency by CD19-cre.

Figure 1—figure supplement 1.

(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 hybrid immunoprecipitation (DRIP) assay in resting B cells. DRIP assay was performed with S9.6 antibody using primary resting B cells; RNase H treatment of Setx-/-Rnaseh2bf/f sample was used as the negative control. Relative enrichment was calculated as chromatin immunoprecipitation (ChIP)/input, and the results were replicated in three independent experiments. Error bars show standard deviation; statistical analysis was performed using one-way ANOVA (n = 4 mice/genotype). (D) Real-time RT-PCR analysis for germline transcripts (Ix-Cx) at donor and acceptor switch regions in WT, Setx-/-, Rnaseh2bf/f, and Setx-/- Rnaseh2bf/f splenic B lymphocytes cultured for 72 hr with LPS/IL-4/α-RP105 stimulation. Expression is normalized to CD79b and is presented as relative to expression in WT cells, set as 1. Error bars show standard deviation; statistical analysis was performed using multiple t-test (n = 4 mice/genotype).
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.
Figure 1—figure supplement 1—source data 2. Numerical data used to generate graphs in Figure 1—figure supplement 1A–D.
Figure 1—figure supplement 2. DNA:RNA hybrid formation at the Actb locus.

Figure 1—figure supplement 2.

(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 primers used in DRIP-qPCR shown in (B) are marked with black (gene body) and pink (3′UTR) triangles. (B) DRIP-qPCR along Actb locus in mouse primary B cells. Diagram of the IgH locus showing the location of PCR products used for DNA:RNA hybrid immunoprecipitation (DRIP) (top panel). DRIP-qPCR along Actb locus in mouse primary B cells (lower panel). DRIP assay was performed with S9.6 antibody using primary B cells after 72 hr of stimulation to IgG1 with LPS/IL-4/α-RP105; RNase H treatment of Setx-/-Rnaseh2bf/f sample was used as the negative control. Relative enrichment was calculated as chromatin immunoprecipitation (ChIP)/input, and the results were replicated in three independent experiments. Error bars show standard deviation; statistical analysis was performed using one-way ANOVA (n = 3 mice/genotype).
Figure 1—figure supplement 2—source data 1. Numerical data used to generate graph in Figure 1—figure supplement 2B.