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. 2014 May 1;10(5):e1004318.
doi: 10.1371/journal.pgen.1004318. eCollection 2014 May.

R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome

Matthias Groh  1 , Michele M P Lufino  2 , Richard Wade-Martins  2 , Natalia Gromak  1
Affiliations

R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome

Matthias Groh et al. PLoS Genet. .

Abstract

Friedreich ataxia (FRDA) and Fragile X syndrome (FXS) are among 40 diseases associated with expansion of repeated sequences (TREDs). Although their molecular pathology is not well understood, formation of repressive chromatin and unusual DNA structures over repeat regions were proposed to play a role. Our study now shows that RNA/DNA hybrids (R-loops) form in patient cells on expanded repeats of endogenous FXN and FMR1 genes, associated with FRDA and FXS. These transcription-dependent R-loops are stable, co-localise with repressive H3K9me2 chromatin mark and impede RNA Polymerase II transcription in patient cells. We investigated the interplay between repressive chromatin marks and R-loops on the FXN gene. We show that decrease in repressive H3K9me2 chromatin mark has no effect on R-loop levels. Importantly, increasing R-loop levels by treatment with DNA topoisomerase inhibitor camptothecin leads to up-regulation of repressive chromatin marks, resulting in FXN transcriptional silencing. This provides a direct molecular link between R-loops and the pathology of TREDs, suggesting that R-loops act as an initial trigger to promote FXN and FMR1 silencing. Thus R-loops represent a common feature of nucleotide expansion disorders and provide a new target for therapeutic interventions.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. R-loops are formed over expanded repeats of FXN gene in FRDA cells.
A. Diagram of FXN gene. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA)n expansion. TSS2 is the major transcriptional start site in lymphoblastoid cells. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS2 in kilobases. B. Cell lines used in the study. The repeat sizes are indicated. C. RT-qPCR analysis of γ-actin, β-actin, GAPDH and FXN mRNAs in control (GM15851) and FRDA (GM15850) cells. Values are normalised to 5S rRNA and relative to control cells. D. RNA Pol II ChIP in control (GM15851) and FRDA (GM15850) cells. E. RT-qPCR analysis of FXN nascent RNA in control (GM15851) and FRDA (GM15850) cells, normalised to 5S rRNA and relative to ex1 RNA in control cells. F. DIP on endogenous FXN gene in control (GM15851) and FRDA (GM15851) cells. γ-actin is positive control. G. R-loops are sensitive to RNase H digestion. DIP samples were treated with 25 U of recombinant E.coli RNase H (NEB, M0297S) for 6 hours at 37°C. γ-actin is positive control. Bars in C–G are average values +/− SEM (n>3).
Figure 2
Figure 2. R-loops are stable and impede Pol II transcription on FXN gene.
A. RT-qPCR analysis of nascent γ-actin and FXN RNA from control and FRDA cells treated with 5 µg/ml of actinomycin D for 21 hours. Values are relative to untreated control cells. B. DIP on FXN gene in control and FRDA cells treated with 5 µg/ml of actinomycin D for 21 hours. γ-actin is positive control. C. H3K9me2 ChIP in control and FRDA cells. H3K9me2 levels were normalized to the total H3 levels. γ-actin is used as background control. D. Diagram depicting the Br-UTP nuclear run-on (NRO) method. E. Br-UTP nuclear run-on in two control (GM15851, GM14926) and two FRDA (GM15850 and GM16243) cells, normalised to the region B in control cells. Bars in A–C and E are average values +/− SEM (n>3).
Figure 3
Figure 3. Over-expressed RNase H1 resolves R-loops formed on FXN expanded repeats in HEK293 cells.
A. Diagram of the FXN-Luc gene, containing 6 (FXN-Luc) or 310 GAA repeats (FXN-GAA-Luc), integrated on the chromosome 1 of HEK293 cells. Frataxin gene was fused to the luciferase at the beginning of the FXN exon 5. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA)n expansion. TSS is the transcriptional start site. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS in kilobases. B. Size of GAA expansion determined by PCR analysis on genomic DNA from FXN-Luc and FXN-GAA-Luc cell lines, using GAA104F and GAA629R primers. PCR products were run on 1% agarose gel. M denotes the marker lane. FXN-Luc and FXN-GAA-Luc cells contain endogenous wild type FXN gene , giving rise to the PCR product of 0.5 kb. C. FXN and γ-actin nascent RNA levels in FXN-Luc (white bars) and FXN-GAA-Luc (black bars) HEK293 cells, determined by RT-qPCR and normalised to 5S rRNA. The level of FXN and γ-actin nascent RNA in FXN-Luc cells was taken as 1. LucR primer was used for the reverse transcription reaction. qPCR was carried using in4F and ex5R primers, shown in A. D. DIP analysis on FXN-Luc gene in FXN-Luc (white bars) and FXN-GAA-Luc (black bars) HEK293 cells using RNA/DNA hybrid-specific S9.6 antibody. E. RT-qPCR analysis of RNase H1 mRNA from FXN-Luc and FXN-GAA-Luc cells, treated with control and RNase H1 siRNAs. Values are normalised to GAPDH mRNA and are relative to FXN-Luc cells, treated with control siRNA. F. DIP analysis on FXN-Luc gene in FXN-Luc and FXN-GAA-Luc HEK293 cells, treated with control and RNase H1 siRNAs. G. Western blot analysis of 50 µg of protein extracts obtained from FXN-Luc and FXN-GAA-Luc cells transfected with Flag and RNase H1-Flag expression plasmids. Western blot was probed with anti-RNase H1 antibody. * denotes endogenous RNase H1 protein. H. DIP analysis on FXN-Luc gene in FXN-Luc and FXN-GAA-Luc HEK293 cells transfected with Flag or RNase H1-Flag expression plasmids. Bars in C–F and H represent the average values from at least three independent experiments +/− SEM.
Figure 4
Figure 4. R-loops are not affected by changes in H3K9 dimethylation.
A. H3K9me2 ChIP in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. H3K9me2 levels were normalized to the total H3 levels and relative to amplicon FXN A, not affected by the treatment. B. DIP analysis in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. C. RT-qPCR analysis of FXN nascent RNA in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. Values are relative to untreated control cells and normalized to γ-actin nascent RNA. Bars in A–C are average values +/− SEM (n>3).
Figure 5
Figure 5. R-loops trigger transcriptional repression of FXN gene.
A. DIP analysis on FXN gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. B. H3K9me2 ChIP on FXN gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. H3K9me2 levels were normalized to the total H3 levels. C. RT-qPCR analysis of FXN nascent RNA in control and FRDA cells, treated with 10 µM camptothecin for 6 hours. Values are relative to untreated control cells and normalized to γ-actin nascent RNA. D. G9a ChIP on FXN gene in control and FRDA cells. G9a levels are normalised relative to amplicon B in control cells. E. Western blot analysis of 20 and 40 µg of protein extracts obtained from FXN-Luc and FXN-GAA-Luc cells, treated with control and Top1 siRNAs. Western blot was probed with anti-Top1 and anti-actin antibody. F. DIP analysis on FXN gene in FXN-Luc and FXN-GAA-Luc HEK293 cells, treated with control and Top1 siRNAs. Bars in A–D and F are average values +/− SEM (n>3).
Figure 6
Figure 6. R-loops are formed over (CGG)n expanded repeats of FMR1 gene.
A. Diagram of FMR1 gene. Black boxes are exons, white box is 5′ UTR and lines are introns. Red triangle is (CGG)n expansion. qPCR amplicons are shown below the diagram. TSS is the transcriptional start site. Numbers indicate the distances to TSS in kilobases. B. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC for 7 days, normalized to GAPDH. C. DIP analysis on endogenous FMR1 gene in control and FXS cells, treated with 1 µM 5-azadC for 7 days. Values are relative to ex1 region in control untreated cells. D. FMR1 R-loops are sensitive to RNase H digestion, following the treatment with 25 U of RNase H for 6 hours at 37°C prior to immuno-precipitation. Values are relative to in15 region in control untreated cells. E. R-loop kinetics on exon 1 of FMR1 gene in control and FXS cells during the process of transcriptional re-activation with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). Values are relative to ex1 region in control untreated cells on day 7. F. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). The level of FMR1 mRNA in control cells is taken as 1. Bars in B–D are average values +/− SEM (n>3).
Figure 7
Figure 7. Model for the role of R-loops in mediating FXN and FMR1 gene silencing.
Background R-loop level on wild type allele allows efficient transcriptional elongation and gene expression. Transcribed (GAA)n and (CGG)n expanded repeats form R-loops resulting in decreased initiation and elongation of RNA Pol II. This leads to downregulation of FXN and FMR1 expression, associated with formation of repressive DNA and chromatin marks.
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