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

Figure 4. 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 subjected to immunoprecipitation and immunoblotting with indicated antibodies. (B) Representative image of alkaline gel from Setx-/-, Setx-/-Rnaseh2bf/f EV, and Setx-/-Rnaseh2bf/f+FLAG-RNaseH2B cells (n = 3 independent mice/genotype). (C) Densitometry trace of representative alkaline gel in (B). (D) DNA:RNA hybrid immunoprecipitation (DRIP) assay was performed with S9.6 antibody on Setx-/-, Setx-/-Rnaseh2bf/f, and Setx-/-Rnaseh2bf/f+FLAG-RNaseH2B-expressing cells stimulated with LPS/IL-4/α-RP105 for 96 hr. RNase H treatment of Setx-/-Rnaseh2bf/f sample was a 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. (E) ChIP analysis for FLAG-RNaseH2B occupancy in Eμ, Sμ, and Sγ regions of primary B cells in response to LPS/IL-4/α-RP105 stimulation. Relative enrichment was calculated as ChIP/input. Error bars show standard deviation. Statistical analysis was performed using Student’s t-test (n = 3 mice/genotype).

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.
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.
Figure 4—source data 3. Numerical data used to generate graphs in Figure 4D and E.

Figure 4.

Figure 4—figure supplement 1. Independent repeats of FLAG-RNaseH2B chromatin immunoprecipitation (ChIP).

Figure 4—figure supplement 1.

Separate graphs for the four independent repeats of the FLAG-RNaseH2B ChIP shown in in Figure 4E.
Figure 4—figure supplement 1—source data 1. Numerical data used to generate graphs in Figure 4—figure supplement 1.