Figure 1.
Evidence of DNA/RNA hybrids (R-loops) accumulation in spermatocytes of ARCA mouse model.
A. Histological cross-sections of seminiferous tubules from ARCA mouse models Setx−/−, Atm−/−, Aptx−/−, Tdp1−/− and wildtype (WT) littermates. Sections were H & E stained. Scale bar, 100 µm. B. Immunostaining of testes sections with R-loop (S9.6) antibody (red) and TUNEL (green). Nuclei were stained using Hoechst 33342 (blue). Scale bar, 100 µm. Magnified views of the tubules are also shown. Representative spermatocytes staining positive for both R-loop and TUNEL are indicated by white arrows. C. Immunostaining of testes sections with γH2AX (red), a marker of DNA DSBs, and TUNEL (green). Scale bar, 100 µm.
Figure 2.
R-loop formation, apoptosis, and DNA damage quantitation in ARCAs mouse testes.
A. Percentage of seminiferous tubules containing R-loops. B. Average number of apoptotic spermatocytes per tubule in testes from ARCA mice. C. Percentage of R-loop & TUNEL-positive cells. D. Average number of apoptotic cells containing DNA DSBs (γH2AX). For each panel, 200 tubules were examined per animal and cells were counted in each tubule (n = 3). Error bars represent SEM.
Figure 3.
Absence of R-loops in Setx−/− nervous tissues.
A. Whole brain sections from wildtype (Setx+/+) and Setx−/− were stained for R-loop (Red) and apoptosis (TUNEL, Green). Nuclei were labelled with DAPI. B. Wildtype and Setx−/− cerebellar sections staining with R-loops and TUNEL. DAPI stained nuclei. C. As a control for R-loop and TUNEL staining, both wildtype and Setx−/− testes sections are shown. Scale bar, 100 µm.
Figure 4.
R-loops forming sequences (RLFS) prediction and R-loops detection using DRIP assay.)
UCSC Genome browser shows chromosome mapping of RLFS and poly(A) signal location at 3′ UTR of studied genes. RFLS (red box), and PCR products (black vertical lines connected by horizontal lines) are indicated on the diagram. A. RLFS prediction in the 3′ UTR of the mouse Pkd2l1 gene located in the HS44.2 locus on chromosome 19. See Figure S5 for sequence details. DRIP quantitation from Setx+/+ and Setx−/− showed similar background levels of R-loops in brain and cerebellum. Only a significant increase in R-loop formation was observed in Setx−/−testes as compared to Setx+/+(Students t-test, p<0.05, n = 3). B. RLFS prediction in the 3′UTR of the mouse Foxo4 gene located on the X chromosome. DRIP quantitation from Setx+/+ and Setx−/− showed similar background levels of R-loops in brain and cerebellum. Similarly, a significant increase in R-loop formation was only observed in Setx−/−testes (Students t-test, p<0.05, n = 3). C. Treatment of Setx−/− DRIP samples with S1 Nuclease and RNAse H confirmed the specificity of the S9.6 (R-loop) antibody towards DNA/RNA hybrids as shown by reduced fluorescence intensities on Bioanalyzer Spectra.
Figure 5.
Induction of R-loop after DNA damage exposure.
A. Knock down of senataxin in HeLa cells using short interfering RNA (siRNA) induced the accumulation of R-loops in nucleoli. Knock down efficiency for senataxin is shown in Figure S2. Treatment of these cells with 25 µM of camptothecin (CPT) led to the accumulation of R-loops in control cells (Ctrl siRNA) and a further increase in senataxin knockdown cells (SETX siRNA). Pre-treatment of these with RNA polymerase inhibitor Actinomycin D (AD, 5 µg/ml), ablated the CPT-induced formation of R-loop in both cell types. B. Quantitation of R-loops formation in control and senataxin knockdown cells. No R-loops were detected in ctrl siRNA-treated cells under normal growing conditions. CPT induced the formation of R-loops in both cell types and pre-treatment these cells with AD prevented the induction of R-loops following DNA damage exposure. Levels of R-loops returned to basal levels in SETX siRNA-treated cells after addition of AD.
Figure 6.
Treatment of Setx and Tdp1 mice with Topotecan induces severe weight loss.
Wiltype (WT), Setx−/− and Tdp1−/− mice were treated with a daily dose of topotecan (TPT, 2 mg/kg/day) over a period of 9 days in order to exacerbate the formation of R-loops in these mice. Mice of each genotype were injected daily with a dose of TPT (2 mg/kg/day) and weights were recorded daily (n = 3). Controls were injected with an equivalent volume of purified water.
Figure 7.
Topotecan treatment does not induce R-loops formation in post-mitotic nervous tissues in Setx−/− mice.
Histological sections of brain, cerebellum, and testes were immunostained for R-loops (Red) and TUNEL (Green). No R-loops were detected in brain and cerebellum sections after TPT treatment. In contrast, an increase in the number of R-loop-containing cells was observed in wildtype and Setx−/− after TPT exposure suggesting that R-loop preferentially form in proliferating cells, most likely due to collision between the DNA replication machinery and the transcription apparatus. DAPI stained nuclei and Ki67 (Red) was used as a marker for proliferation. Scale bar, 100 µm.
Figure 8.
Induction of R-loops in proliferating cells after topotecan treatment.
Wildtype and Setx−/−histological sections of intestine and spleen, two tissues with a high proliferative capacity, were stained for R-loops, TUNEL, and Ki67. TPT induced the formation of R-loops and apoptosis in both intestine and spleen. TPT disrupted the structure of the small intestine in Setx−/− animals supporting the severe weight loss observed in these mice. R-loop (red), TUNEL (green) and Ki67 (red). DAPI (blue) stained nuclei. Scale bar, 100 µm.
Figure 9.
Induction of R-loops only in proliferative tissues in Tdp1−/− after topotecan treatment.
Histological sections from Tdp1−/− mice were stained for R-loops, TUNEL and Ki67 after topotecan exposure. Similar to Setx−/−, TPT induced the formation of R-loops formation only in proliferative tissues testes, intestine and spleen). No R-loops were detected in post-mitotic tissues (brain and cerebellum). Scale bar, 100 µm.
Figure 10.
Increased R-loop formation in TPT-treated knockout Setx and Tdp1 mice.
Graphs show the percentage of apoptotic (TUNEL) cells only, cells containing R-loops only and apoptotic cells containing R-loops in the different tissues of Setx (A and B) and Tdp1 mice (C). Counts were performed using 5 fields of view per tissue per animal (n = 3). Higher levels of these three cell types were observed in TPT-treated knockout animals.
Figure 11.
R-loops preferentially form in cycling but not in post-mitotic cells.
Model depicting the requirements for R-loops formation in the various ARCAs model. While transcription is implied in the formation of R-loops, active replication appears to be a necessary condition too. Thus R-loops accumulation does not appear to contribute to the neurodegenerative phenotype observed in autosomal recessive cerebellar ataxias.