History of R-loop research.

<p>The diagram depicts major developments in the R-loop field and diseases associated with R-loop dysregulation.</p

Potential R-loop-based therapeutic approach in Angelman Syndrome (AS).

<p><b>A.</b> Neuronal expression of the paternal ncRNA <i>Ube3a-ATS</i> represses paternal <i>Ube3a</i> gene in cis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Meng1" target="_blank">[101]</a>. DNA methylation of the <i>Snord116</i> locus on the maternal allele prevents <i>Ube3a-ATS</i> transcription, resulting in <i>Ube3a</i> expression from the maternal allele. Transcriptional repression is indicated by red crosses. <b>B.</b> R-loop-mediated re-activation of silent paternal <i>Ube3a gene</i> provides a targeted therapy for AS. Deletion leading to the loss of maternal <i>Ube3a</i> expression detected in AS is indicated by the red dashed line. Topotecan treatment increases R-loop levels over the <i>Snord116</i> locus, resulting in chromatin decondensation, inhibition of Pol II transcription through <i>Ube3a-ATS</i>, and increased expression of <i>Ube3a</i> from the paternal allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Powell1" target="_blank">[50]</a>.</p

R-loops and human diseases.

<p>The diagram depicts the role of R-loops in human diseases. Loss of wild type protein function is depicted by red crosses. <b>A.</b> Ataxia and motor neuron diseases. Mutations in human RNA/DNA helicase senataxin are associated with AOA2/ALS4 disorders and lead to R-loop accumulation and defects in transcriptional termination by Pol II <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-SkourtiStathaki2" target="_blank">[16]</a>, the maintenance of genome integrity <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Yuce1" target="_blank">[46]</a>, meiotic recombination during spermatogenesis, gene silencing during meiotic sex chromosome inactivation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Becherel1" target="_blank">[14]</a>, and neuronal differentiation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Vantaggiato1" target="_blank">[49]</a>. <b>B.</b> Aicardi-Goutières syndrome (AGS). AGS is associated with mutations in all three subunits of RNase H2, ssDNA 3′–5′ exonuclease TREX1 (DNASEIII), dsRNA-editing enzyme ADAR1, and dNTP triphosphatase SAMHD1; these trigger accumulation of unprocessed nucleic acids, including genomic DNA with incorporated ribonucleotides, R-loops, and retroelement-derived nucleic acids, and result in the immune response characteristic of AGS <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Rabe1" target="_blank">[65]</a>. <b>C.</b> Trinucleotide expansion diseases. R-loops form over expanded repeats and result in decreased initiation and elongation of RNA Pol II and formation of repressive chromatin marks, which silence the host gene containing expanded repeats <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Groh1" target="_blank">[75]</a>. <b>D.</b> Genome instability in cancer. Loss of proteins protecting against abnormal R-loop accumulation, such as FIP1L1, leads to genome instability, one hallmark of cancer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Stirling1" target="_blank">[31]</a>. Yellow stars denote double-stranded DNA breaks. <b>E.</b> AID-mediated mutagenesis and translocations in cancer. Single-stranded DNA in R-loops is a substrate for cytidine deamination by activation-induced cytidine deaminase, leading to mutagenesis as indicated by orange stars <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Yu1" target="_blank">[21]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Ruiz1" target="_blank">[88]</a>. These mutations can cause DSB formation, leading to chromosomal translocations. The <i>IgH/c-MYC</i> translocation brings the strong <i>IgH</i> enhancers, shown as yellow box, close to <i>c-MYC</i>, leading to its overexpression in Burkitt's lymphoma <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Robbiani1" target="_blank">[87]</a>. Transcription of <i>IgH/c-MYC</i> starts from a previously inactive promoter downstream of the translocation break point. The <i>IgH</i> locus is depicted in blue, <i>c-MYC</i> gene is in grey. The translocation breakpoint is indicated by a dashed black line. <b>F.</b> Senescence. R-loops formed by the noncoding RNA TERRA accumulate at telomeres in cells deficient of Hpr1 and RNase H. In the absence of telomerase, these R-loops promote Rad52-dependent telomere elongation and delayed senescence. In the absence of telomerase and Rad52, R-loops promote telomere shortening and premature senescence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004630#pgen.1004630-Balk1" target="_blank">[94]</a>.</p

Atroposelective Heck Macrocyclization: Enantioselective Synthesis of Bis(bibenzylic) Natural Products

The Heck protocol was applied for the first time to the atroposelective synthesis of macrocyclic natural products. As ring closure to bis(bibenzyls) of the isoplagiochin type leads to a configurationally stable biaryl axis in the molecule, cyclization could be conducted atroposelectively in the presence of a chiral BINAP ligand

R-loops are not affected by changes in H3K9 dimethylation.

<p>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 <i>FXN</i> 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 <i>FXN</i> 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).</p

R-loops are stable and impede Pol II transcription on <i>FXN</i> gene.

<p>A. RT-qPCR analysis of nascent γ-actin and <i>FXN</i> 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 <i>FXN</i> 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).</p

Model for the role of R-loops in mediating <i>FXN</i> and <i>FMR1</i> gene silencing.

<p>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 <i>FXN</i> and <i>FMR1</i> expression, associated with formation of repressive DNA and chromatin marks.</p

R-loops are formed over expanded repeats of <i>FXN</i> gene in FRDA cells.

<p>A. Diagram of <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>E.coli</i> 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).</p

R-loops trigger transcriptional repression of <i>FXN</i> gene.

<p>A. DIP analysis on <i>FXN</i> gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. B. H3K9me2 ChIP on <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells, treated with control and Top1 siRNAs. Western blot was probed with anti-Top1 and anti-actin antibody. F. DIP analysis on <i>FXN</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> HEK293 cells, treated with control and Top1 siRNAs. Bars in A–D and F are average values +/− SEM (n>3).</p

Over-expressed RNase H1 resolves R-loops formed on <i>FXN</i> expanded repeats in HEK293 cells.

<p>A. Diagram of the FXN-Luc gene, containing 6 (<i>FXN-Luc</i>) or 310 GAA repeats (<i>FXN-GAA-Luc</i>), integrated on the chromosome 1 of HEK293 cells. Frataxin gene was fused to the luciferase at the beginning of the <i>FXN</i> 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 <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cell lines, using GAA104F and GAA629R primers. PCR products were run on 1% agarose gel. M denotes the marker lane. <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells contain endogenous wild type <i>FXN</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004318#pgen.1004318-Lufino1" target="_blank">[23]</a>, giving rise to the PCR product of 0.5 kb. C. <i>FXN</i> and γ-actin nascent RNA levels in <i>FXN-Luc</i> (white bars) and <i>FXN-GAA-Luc</i> (black bars) HEK293 cells, determined by RT-qPCR and normalised to 5S rRNA. The level of <i>FXN</i> and γ-actin nascent RNA in <i>FXN-Luc</i> 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 <i>FXN-Luc</i> gene in <i>FXN-Luc</i> (white bars) and <i>FXN-GAA-Luc</i> (black bars) HEK293 cells using RNA/DNA hybrid-specific S9.6 antibody. E. RT-qPCR analysis of RNase H1 mRNA from <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells, treated with control and RNase H1 siRNAs. Values are normalised to GAPDH mRNA and are relative to <i>FXN-Luc</i> cells, treated with control siRNA. F. DIP analysis on <i>FXN-Luc</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> HEK293 cells, treated with control and RNase H1 siRNAs. G. Western blot analysis of 50 µg of protein extracts obtained from <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> 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 <i>FXN-Luc</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> 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.</p