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. 2015 Nov 19;11(11):e1005674.
doi: 10.1371/journal.pgen.1005674. eCollection 2015 Nov.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops

María L García-Rubio  1 , Carmen Pérez-Calero  1 , Sonia I Barroso  1 , Emanuela Tumini  1 , Emilia Herrera-Moyano  1 , Iván V Rosado  2 , Andrés Aguilera  1
Affiliations

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops

María L García-Rubio et al. PLoS Genet. .

Abstract

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. R loop accumulation in Fanconi Anemia-deficient patient cells.
(A) DRIP-qPCR using the anti-RNA-DNA hybrids S9.6 monoclonal antibody, in FANCA-deficient human HSC72 lymphocytes and the corrected FANCA+ cells at APOE, RPL13A, EGR1, and BTBD19 genes. Pre-immunoprecipitated samples were untreated (-) or treated (+) with RNase H (RNH) as indicated. Signal values of RNA-DNA hybrids immunoprecipitated in each region, normalized to input values and to the signal at the SNRPN negative control region are shown. Data represent mean ± SEM from three independent experiments. *, P < 0.05 (Mann-Whitney U test). (B) Relative amount of R loops in patient FANCD2-/- human PD20 cell line and the corrected PD20 FANCD2+/+ control at 4 different genes. Details as in (A). (C) Levels of DNA-RNA hybrids accumulated in actively transcribed genes in HeLa + siC, HeLa + siFANCD2 cells, as determined by DRIP-qPCR using the S9.6 monoclonal antibody with and without RNase H (RNH) treatment. Details as in (A).
Fig 2
Fig 2. RNA-DNA hybrid accumulation in FANCD2-depleted HeLa cells.
Immunostaining with S9.6 and nucleolin antibodies of siC and siFANCD2 HeLa cells. The graph shows the median of the S9.6 signal intensity per nucleus after nucleolar signal removal. More than 300 cells from two independent experiments were considered. ***, P < 0.001 (Mann-Whitney U test, two-tailed).
Fig 3
Fig 3. R loops in FANCD2-deficient murine cells.
(A) Relative levels of RNA-DNA hybrids as determined by DRIP-qPCR in the Acat3 gene at 3 independent regions in WT and FANCD2-/- MEFs with and without RNase H (RNH) treatment. (B) Relative levels of R loops as detected by DRIP-qPCR analysis at independent regions of the Acat3 gene in WT and FANCD2-/- murine bone marrow Gr1+ cells with and without RNase H treatment. (C) Relative levels of R loops as detected by DRIP-qPCR analysis at independent regions of the Acat3 gene in wild-type and FANCD2-/- murine bone marrow B220 cells with and without RNase H (RNH) treatment. Other details as in Fig 1.
Fig 4
Fig 4. Genome instability in FANCD2-depleted human cells.
(A) Detection of γH2AX foci by IF in siC and siFANCD2 HeLa cells transfected with pcDNA3 (-RNH1) or pcDNA3-RNaseH1 (+RNH1) for RNase H1 overexpression and either untreated or treated for 16 h with 80 ng/ml mitomycin C (MMC). Nuclei were stained with DAPI. The graph shows the quantification of the relative amount of cells containing >10 γH2AX foci with respect to the siC in each case. More than 100 cells overexpressing RNase H1 (positive-stained) or more than 100 cells of mixed population transfected with the empty vector were counted in each of the three experiments. Data represent mean ± SEM from three independent experiments. The red asterisks refer to the comparison of each MMC-treated samples versus its own untreated sample. *, P < 0.05 (Mann-Whitney U test). As a reference the percentage of cells with ≥10 γH2AX foci is >75% in siC cells treated with MMC (B) DNA breaks measured by single-cell gel electrophoresis (comet assay) of siC and siFANCD2 HeLa cells treated or untreated for 4 h with 50 μM cordycepin. The graph shows the median comet tail moment. More than 100 cells were counted in each of the three experiments. Data represent mean ± SEM from three independent experiments. *, P < 0.05 (Student’s t-test).
Fig 5
Fig 5. FANCD2 foci assemble in a R-loop dependent manner.
(A) Immunofluorescence of FANCD2 in HeLa cells with or without RNase H1 overexpression treated or untreated for 16 h with 40 ng/ml MMC and then released for 9 h. More than 100 cells overexpressing RNase H1 (positive-stained) or more than 100 cells of mixed population transfected with the empty vector were counted in each of the three experiments. The graph shows the quantification of the percentage of cells. containing >5 FANCD2 foci. Data represent mean ± SEM from three independent experiments. *, P < 0.05 (Student’s t-test). **, P < 0.01 (Student’s t-test). Other details as in Fig 4. (B) ChIP analysis of FANCA recruitment in HeLa cells with or without RNase H1 overexpression. Signal values of DNA immunoprecipitated in each region, normalized to input values and to the signal without antibody are shown. Data represent mean ± SEM from four independent experiments. *, P < 0.05 (Mann-Whitney U test). (C) ChIP analysis of γH2AX in siC and siFANCD2 HeLa cells with or without RNase H1 overexpression. Data represent mean ± SEM from four independent experiments. *, P < 0.05 (Mann-Whitney U test).
Fig 6
Fig 6. Model for a role of the FA pathway in preventing R-loop accumulation.
The model explains the role of the FA pathway in preventing R loop-mediated genome instability. The FA pathway prevents R loop accumulation that hampers replication fork (RF) progression and leads to DNA breaks. For clarity, only the MCM helicase is depicted at the replication fork.
See this image and copyright information in PMC

References

    1. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 2005; 434: 864–70 - PubMed
    1. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature. 2005; 434: 907–13 - PubMed
    1. Aguilera A, Garcia-Muse T. Causes of genome instability. Annu Rev Genet. 2013; 47: 1–32 10.1146/annurev-genet-111212-133232 - DOI - PubMed
    1. Aguilera A, Garcia-Muse T. R loops: from transcription byproducts to threats to genome stability. Molecular cell. 2012; 46: 115–24 10.1016/j.molcel.2012.04.009 - DOI - PubMed
    1. Skourti-Stathaki K, Kamieniarz-Gdula K, Proudfoot NJ. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature. 2014; 516: 436–9 10.1038/nature13787 - DOI - PMC - PubMed

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