R Loops: From Physiological to Pathological Roles

DNA-RNA hybrids play a physiological role in cellular processes, but often, they represent non-scheduled co-transcriptional structures with a negative impact on transcription, replication and DNA repair. Accumulating evidence suggests that they constitute a source of replication stress, DNA breaks and genome instability. Reciprocally, DNA breaks facilitate DNA-RNA hybrid formation by releasing the double helix torsional conformation. Cells avoid DNA-RNA accumulation by either preventing or removing hybrids directly or by DNA repair-coupled mechanisms. Given the R-loop impact on chromatin and genome organization and its potential relation with genetic diseases, we review R-loop homeostasis as well as their physiological and pathological roles. DNA-RNA hybrids play a physiological role in cellular processes, but often, they represent non-scheduled co-transcriptional structures with a negative impact on transcription, replication and DNA repair. Accumulating evidence suggests that they constitute a source of replication stress, DNA breaks and genome instability. Reciprocally, DNA breaks facilitate DNA-RNA hybrid formation by releasing the double helix torsional conformation. Cells avoid DNA-RNA accumulation by either preventing or removing hybrids directly or by DNA repair-coupled mechanisms. Given the R-loop impact on chromatin and genome organization and its potential relation with genetic diseases, we review R-loop homeostasis as well as their physiological and pathological roles. R loops are non-B DNA structures that form during transcription when the nascent RNA anneals with the template DNA strand, thus displacing the non-template DNA strand. The term R loop refers to the three-stranded structure formed by the DNA-RNA hybrid and the displaced single-stranded DNA (ssDNA), whereas DNA-RNA hybrid only refers to the double-stranded structure—even though, in many reports, both terms are used indistinctly. Generally, R loops form behind elongating RNA polymerases as structures that can be longer than 1 kb (Garcia-Pichardo et al., 2017Garcia-Pichardo D. Canas J.C. Garcia-Rubio M.L. Gomez-Gonzalez B. Rondon A.G. Aguilera A. Histone Mutants Separate R Loop Formation from Genome Instability Induction.Mol. Cell. 2017; 66: 597-609Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Li and Manley, 2005Li X. Manley J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.Cell. 2005; 122: 365-378Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, Yu et al., 2003Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Crossref PubMed Scopus (455) Google Scholar). There are two types of R loops: physiological and pathological. Physiological R loops normally rely on a programmed process requiring specific factors that warrant their formation; pathological R loops occur accidentally in a non-scheduled manner. DNA-RNA hybrid formation is specifically enhanced at certain regions where they play a physiological function. These include Immunoglobulin class switching recombination of B cells in vertebrates, mitochondrial DNA replication, CRISPR-Cas9 gene editing, specific regulatory steps in transcription initiation and termination, telomere homeostasis, and some cases of bacterial plasmid replication among others (Aguilera and García-Muse, 2012Aguilera A. García-Muse T. R loops: from transcription byproducts to threats to genome stability.Mol. Cell. 2012; 46: 115-124Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, Crossley et al., 2019Crossley M.P. Bocek M. Cimprich K.A. R-Loops as Cellular Regulators and Genomic Threats.Mol. Cell. 2019; 73: 398-411Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, hybrids have also been detected all over the genome of bacterial and eukaryotic cells. The frequency at which these occur at every transcribed unit has not been established yet, but it is likely too low to represent a physiological structure with a global functional role, but rather an accidental structure with a negative impact in the cell. In general, R loops may interfere with DNA replication, repair, and transcription, thus compromising genome integrity and function. Therefore, cells have developed different mechanisms to either prevent or resolve such DNA-RNA hybrids. When any of these mechanisms fail, R loops become a threat to genome integrity and cell proliferation, becoming a potential source of cellular pathologies. Apart from the fact that an ssDNA may be highly accessible to metabolites, reactive oxygen species (ROS), DNA modifying enzymes, or nucleases that would increase the incidence of DNA damage (Lindahl, 1993Lindahl T. Instability and decay of the primary structure of DNA.Nature. 1993; 362: 709-715Crossref PubMed Scopus (3734) Google Scholar) features that can apply to the displaced ssDNA of an R loop, accumulated evidence suggests that R-loop-mediated replication fork (RF) stalling is a major feature of transcription-replication conflicts and R-loop-induced DNA damage (Boubakri et al., 2010Boubakri H. de Septenville A.L. Viguera E. Michel B. The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo.EMBO J. 2010; 29: 145-157Crossref PubMed Scopus (151) Google Scholar, Tuduri et al., 2009Tuduri S. Crabbé L. Conti C. Tourrière H. Holtgreve-Grez H. Jauch A. Pantesco V. De Vos J. Thomas A. Theillet C. et al.Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription.Nat. Cell Biol. 2009; 11: 1315-1324Crossref PubMed Scopus (274) Google Scholar, Wellinger et al., 2006Wellinger R.E. Prado F. Aguilera A. Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex.Mol. Cell. Biol. 2006; 26: 3327-3334Crossref PubMed Scopus (101) Google Scholar). In order to understand the impact of R loops in cell fate and proliferation, we need to answer questions such as what differentiates physiological from unscheduled pathological R loops, how often unscheduled R loops form along genomes, how cells protect themselves from pathological R loops, or how R loops can affect chromatin structure and compromise genome integrity. Here, we review the relevant data that have garnered attention for R loops in the contexts of transcription, replication, and repair, with the aim of providing a current view of the molecular mechanisms controlling R-loop homeostasis and how this might relate to disease. Research on R-loop characteristics and distribution along the genome depends on the tools available to detect them in vivo. Different methodologies have been developed to detect DNA-RNA hybrids in vivo that include the isolation and physical analysis of nucleic acids resistant to ribonuclease (RNase) A and sensitive to RNase H (Huertas and Aguilera, 2003Huertas P. Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.Mol. Cell. 2003; 12: 711-721Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar); electron microscopy (EM) (Backert, 2002Backert S. R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1.EMBO J. 2002; 21: 3128-3136Crossref PubMed Scopus (20) Google Scholar) or chromatin immunoprecipitation (ChIP) and/or immunofluorescence (IF) using an inactivated RNase H (Chen et al., 2017Chen L. Chen J.Y. Zhang X. Gu Y. Xiao R. Shao C. Tang P. Qian H. Luo D. Li H. et al.R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.Mol. Cell. 2017; 68: 745-757Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) and the RNase H1 hybrid-binding domain (HB) fused to the green fluorescent protein (GFP) (Bhatia et al., 2014Bhatia V. Barroso S.I. García-Rubio M.L. Tumini E. Herrera-Moyano E. Aguilera A. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.Nature. 2014; 511: 362-365Crossref PubMed Scopus (193) Google Scholar) (Figure 1A). Nevertheless, the most commonly used methods rely on the S9.6 anti-10 nucleotide DNA-RNA hybrid monoclonal antibody (Boguslawski et al., 1986Boguslawski S.J. Smith D.E. Michalak M.A. Mickelson K.E. Yehle C.O. Patterson W.L. Carrico R.J. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids.J. Immunol. Methods. 1986; 89: 123-130Crossref PubMed Scopus (159) Google Scholar) (Figure 1A). This antibody has been extensively used for DNA-RNA hybrid immunoprecipitation (DRIP) followed by either qPCR at specific DNA regions or by sequencing for genome-wide studies, as well as for IF. These analyses have enabled a relatively precise idea on the distribution of hybrids along the different regions of eukaryotic genomes, mainly from Saccahromyces cerevisae and human cells (El Hage et al., 2010El Hage A. French S.L. Beyer A.L. Tollervey D. Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis.Genes Dev. 2010; 24: 1546-1558Crossref PubMed Scopus (216) Google Scholar, Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Wahba et al., 2011Wahba L. Amon J.D. Koshland D. Vuica-Ross M. RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability.Mol. Cell. 2011; 44: 978-988Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Sanz et al., 2016Sanz L.A. Hartono S.R. Lim Y.W. Steyaert S. Rajpurkar A. Ginno P.A. Xu X. Chédin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.Mol. Cell. 2016; 63: 167-178Abstract Full Text Full Text PDF PubMed Google Scholar). In both DRIP and IF assays, treatment with RNase H1, which specifically degrades the RNA moiety of the hybrid, is required because the S9.6 antibody is also able to recognize double-stranded RNAs (dsRNAs) present at high concentration at specific sites (Hartono et al., 2018Hartono S.R. Malapert A. Legros P. Bernard P. Chédin F. Vanoosthuyse V. The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast.J. Mol. Biol. 2018; 430: 272-284Crossref PubMed Scopus (24) Google Scholar, Silva et al., 2018Silva S. Camino L.P. Aguilera A. Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability.Proc. Natl. Acad. Sci. USA. 2018; 115: 11024-11029Crossref PubMed Scopus (5) Google Scholar). In general, the initial genome-wide DRIP analyses have been confirmed and extended by other approaches. These include using strand-specific high-throughput sequencing of either DNA libraries, marked with dUTP or with adaptors (Nadel et al., 2015Nadel J. Athanasiadou R. Lemetre C. Wijetunga N.A. Ó Broin P. Sato H. Zhang Z. Jeddeloh J. Montagna C. Golden A. et al.RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships.Epigenetics Chromatin. 2015; 8: 46Crossref PubMed Scopus (49) Google Scholar, Xu et al., 2017Xu W. Xu H. Li K. Fan Y. Liu Y. Yang X. Sun Q. The R-loop is a common chromatin feature of the Arabidopsis genome.Nat. Plants. 2017; 3: 704-714Crossref PubMed Scopus (28) Google Scholar), or cDNA libraries obtained by RT-PCR from the RNA moiety of hybrids after DNase I treatment (Sanz et al., 2016Sanz L.A. Hartono S.R. Lim Y.W. Steyaert S. Rajpurkar A. Ginno P.A. Xu X. Chédin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.Mol. Cell. 2016; 63: 167-178Abstract Full Text Full Text PDF PubMed Google Scholar). Genome-wide analysis have unveiled R loops to be present in normal/wild-type cells at more DNA regions than anticipated. Indeed, genome coverage data oscillate from 8% in yeast to 10% in Arabidopsis or 5% in human cells (Wahba et al., 2016Wahba L. Costantino L. Tan F.J. Zimmer A. Koshland D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.Genes Dev. 2016; 30: 1327-1338Crossref PubMed Google Scholar, Xu et al., 2017Xu W. Xu H. Li K. Fan Y. Liu Y. Yang X. Sun Q. The R-loop is a common chromatin feature of the Arabidopsis genome.Nat. Plants. 2017; 3: 704-714Crossref PubMed Scopus (28) Google Scholar, Sanz et al., 2016Sanz L.A. Hartono S.R. Lim Y.W. Steyaert S. Rajpurkar A. Ginno P.A. Xu X. Chédin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.Mol. Cell. 2016; 63: 167-178Abstract Full Text Full Text PDF PubMed Google Scholar). Altogether the data indicate that DNA-RNA hybrids accumulate preferentially at highly transcribed genes, including the rRNA and tRNA loci, as well as in some transposable elements (Ty in yeast), centromeres and telomeres, antisense-RNAs or ncRNAs regions (Chan et al., 2014Chan Y.A. Aristizabal M.J. Lu P.Y. Luo Z. Hamza A. Kobor M.S. Stirling P.C. Hieter P. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.PLoS Genet. 2014; 10: e1004288Crossref PubMed Scopus (103) Google Scholar, Chen et al., 2017Chen L. Chen J.Y. Zhang X. Gu Y. Xiao R. Shao C. Tang P. Qian H. Luo D. Li H. et al.R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.Mol. Cell. 2017; 68: 745-757Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, El Hage et al., 2014El Hage A. Webb S. Kerr A. Tollervey D. Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria.PLoS genetics. 2014; 10: e1004716Crossref PubMed Scopus (0) Google Scholar, Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Wahba et al., 2016Wahba L. Costantino L. Tan F.J. Zimmer A. Koshland D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.Genes Dev. 2016; 30: 1327-1338Crossref PubMed Google Scholar). In yeast, the R-loop-prone open reading frames (ORFs) are generally highly transcribed and have high GC content, but the proportion of ORFs observed fluctuates from 20% to more than 65% depending on the study (Wahba et al., 2016Wahba L. Costantino L. Tan F.J. Zimmer A. Koshland D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.Genes Dev. 2016; 30: 1327-1338Crossref PubMed Google Scholar, El Hage et al., 2014El Hage A. Webb S. Kerr A. Tollervey D. Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria.PLoS genetics. 2014; 10: e1004716Crossref PubMed Scopus (0) Google Scholar, Chan et al., 2014Chan Y.A. Aristizabal M.J. Lu P.Y. Luo Z. Hamza A. Kobor M.S. Stirling P.C. Hieter P. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.PLoS Genet. 2014; 10: e1004288Crossref PubMed Scopus (103) Google Scholar). This variability may be due to technical differences in the DNA-RNA immunoprecipitation protocols (see Halász et al., 2017Halász L. Karányi Z. Boros-Oláh B. Kuik-Rózsa T. Sipos É. Nagy É. Mosolygó-L Á. Mázló A. Rajnavölgyi É. Halmos G. Székvölgyi L. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases.Genome Res. 2017; 27: 1063-1073Crossref PubMed Scopus (22) Google Scholar). In the case of mammalian cells, R loops are also detected mainly at active genes, and they also accumulate at promoter and terminator regions in which they play a role in gene expression regulation (Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Sanz et al., 2016Sanz L.A. Hartono S.R. Lim Y.W. Steyaert S. Rajpurkar A. Ginno P.A. Xu X. Chédin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.Mol. Cell. 2016; 63: 167-178Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, these studies have permitted the identification of sequences prone to DNA-RNA hybridization such as GC-rich sequences, CpG islands, transcription termination sites with high GC-skew, or G-quadruplex (G4)-containing sequences (Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Sanz et al., 2016Sanz L.A. Hartono S.R. Lim Y.W. Steyaert S. Rajpurkar A. Ginno P.A. Xu X. Chédin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.Mol. Cell. 2016; 63: 167-178Abstract Full Text Full Text PDF PubMed Google Scholar, Chen et al., 2017Chen L. Chen J.Y. Zhang X. Gu Y. Xiao R. Shao C. Tang P. Qian H. Luo D. Li H. et al.R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.Mol. Cell. 2017; 68: 745-757Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Stork et al., 2016Stork C.T. Bocek M. Crossley M.P. Sollier J. Sanz L.A. Chédin F. Swigut T. Cimprich K.A. Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage.eLife. 2016; 5: e17548Crossref PubMed Google Scholar). Despite a general agreement about such features, certain regions were observed using specific protocols. For example, R-loop-prone regions are preferentially AT-rich when detected by DRIP analysis combined with S1 nuclease digestion in yeast (Wahba et al., 2016Wahba L. Costantino L. Tan F.J. Zimmer A. Koshland D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.Genes Dev. 2016; 30: 1327-1338Crossref PubMed Google Scholar). However, in this case, it would be important to exclude the possibility that S1-induced nicks do not favor DNA-RNA annealing during the DRIP protocol. In contrast to DNA-RNA hybrids, R-loop detection relies on the identification of the displaced ssDNA. Several reports have shown that the ssDNA-binding protein RPA binds the displaced ssDNA (Chaudhuri et al., 2004Chaudhuri J. Khuong C. Alt F.W. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets.Nature. 2004; 430: 992-998Crossref PubMed Scopus (298) Google Scholar, Nguyen et al., 2018Nguyen H.D. Leong W.Y. Li W. Reddy P.N.G. Sullivan J.D. Walter M.J. Zou L. Graubert T.A. Spliceosome Mutations Induce R Loop-Associated Sensitivity to ATR Inhibition in Myelodysplastic Syndromes.Cancer Res. 2018; 78: 5363-5374Crossref PubMed Scopus (22) Google Scholar), suggesting that one could use anti-RPA antibodies to detect R loops. However, since RPA binds to ssDNA generated at resected double-strand breaks (DSBs) and at the lagging strand of RFs, it is unlikely that an RPA signal can be unambiguously attributed to R loops. Indirectly, in vivo R loops can be inferred with high precision by the mutation profile generated by sodium bisulfite in single DNA molecules purified from cell cultures (Yu et al., 2003Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Crossref PubMed Scopus (455) Google Scholar) or by the in vivo action of the human activation-induced cytidine deaminase (AID) (Gómez-González and Aguilera, 2007Gómez-González B. Aguilera A. Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex.Proc. Natl. Acad. Sci. USA. 2007; 104: 8409-8414Crossref PubMed Scopus (68) Google Scholar) (Figure 1A). Whereas both have proved to be reliable to detect R loops in vivo (Garcia-Pichardo et al., 2017Garcia-Pichardo D. Canas J.C. Garcia-Rubio M.L. Gomez-Gonzalez B. Rondon A.G. Aguilera A. Histone Mutants Separate R Loop Formation from Genome Instability Induction.Mol. Cell. 2017; 66: 597-609Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), only the bisulfite mutagenesis can provide a measurement of the length of DNA-RNA hybrids because the in vitro treatment by bisulfite induces deamination of all cytidines within DNA, whereas in vivo AID-induced mutation is not as processive. With this method, the mutation profile of the non-template ssDNA region allows the inference of the average length of R loops, provided that such a profile is abolished by RNase H1 treatment. Some estimates have been obtained in human cells and yeast, reporting sizes ranging from less than 100 bp up to 2 kb (Garcia-Pichardo et al., 2017Garcia-Pichardo D. Canas J.C. Garcia-Rubio M.L. Gomez-Gonzalez B. Rondon A.G. Aguilera A. Histone Mutants Separate R Loop Formation from Genome Instability Induction.Mol. Cell. 2017; 66: 597-609Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Li and Manley, 2005Li X. Manley J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.Cell. 2005; 122: 365-378Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, Yu et al., 2003Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Crossref PubMed Scopus (455) Google Scholar). Comparison of the length of the DNA regions identified as R-loop-prone in the available DRIP-seq experiments shows two different behaviors: a narrow-sized distribution (180–2,350 bp) or a wide-sized distribution (180–22,500 bp) depending on the methodology used to break the DNA preparation prior to immunoprecipitation. Sonication yields shorter R-loop-prone DNA segments, while restriction enzymes yields long segments likely due to the more spaced distribution of the DNA restriction sites (Halász et al., 2017Halász L. Karányi Z. Boros-Oláh B. Kuik-Rózsa T. Sipos É. Nagy É. Mosolygó-L Á. Mázló A. Rajnavölgyi É. Halmos G. Székvölgyi L. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases.Genome Res. 2017; 27: 1063-1073Crossref PubMed Scopus (22) Google Scholar). Importantly, with the described methodology, we are yet unable to determine the frequency at which R loops originate, so we do not have a measure of how often R loops occur in cells or at specific DNA regions. However, we can affirm that R loop occurs co-transcriptionally over conserved genomic regions, presumably facing a dynamic turnover. Thus, to understand the physiological meaning and causes of R loop distribution, we must consider the different transcription profiles, the signals and environmental conditions of growth, the cell cycle stage, or the specific epigenetic features of each cell or DNA region among other features. R loops are intermediates required for several processes regulating genome dynamics such as immunoglobulin (Ig) class-switch recombination (CSR) (Yu et al., 2003Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Crossref PubMed Scopus (455) Google Scholar) or CRISPR-Cas9 activity, in which the guide-RNA forms a DNA-RNA hybrid as an intermediate to identify the target for Cas9-mediated cleavage (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (5293) Google Scholar), or the initiation of DNA replication in mitochondrial DNA, bacterial plasmids, and the bacteriophages ColE1 and T4 (see Aguilera and García-Muse, 2012Aguilera A. García-Muse T. R loops: from transcription byproducts to threats to genome stability.Mol. Cell. 2012; 46: 115-124Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar) (Figure 1B). Also, increasing evidence indicates that R loops play roles in gene expression, as suggested by the fact that such structures are enriched in gene promoters and termination regions. DNA-RNA hybrids have been linked to the unmethylated state of CpG islands, which are present at promoters of many genes in mammalian cells. Computational and experimental data suggest that R loops formed at CpG islands protect these regions against DNA methylation, ultimately avoiding transcription silencing (Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Grunseich et al., 2018Grunseich C. Wang I.X. Watts J.A. Burdick J.T. Guber R.D. Zhu Z. Bruzel A. Lanman T. Chen K. Schindler A.B. et al.Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.Mol. Cell. 2018; 69: 426-437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). This protection could be explained by the observation that DNA methyl-transferase 1 preferentially binds and methylates double-stranded DNA over the DNA-RNA hybrid (Grunseich et al., 2018Grunseich C. Wang I.X. Watts J.A. Burdick J.T. Guber R.D. Zhu Z. Bruzel A. Lanman T. Chen K. Schindler A.B. et al.Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.Mol. Cell. 2018; 69: 426-437Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) (Figure 1C). In addition, R loops can guide the binding of transcription regulation factors, as described for the regulation driven by long non-coding RNAs (lncRNAs) (Figure 1C). Thus, in Arabidopsis, the expression of a set of lncRNAs called COOLAIR leads to silencing of the FLC gene. The homeodomain protein AtNDX inhibits the expression of COOLAIR by binding to the ssDNA of an R loop formed at its promoter, ultimately allowing transcription of the FLC gene (Sun et al., 2013Sun Q. Csorba T. Skourti-Stathaki K. Proudfoot N.J. Dean C. R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus.Science. 2013; 340: 619-621Crossref PubMed Scopus (154) Google Scholar). Interestingly, yeast cells also respond to environmental changes with the formation of R loops by lncRNAs, but in this case to activate expression (Cloutier et al., 2016Cloutier S.C. Wang S. Ma W.K. Al Husini N. Dhoondia Z. Ansari A. Pascuzzi P.E. Tran E.J. Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.Mol. Cell. 2016; 61: 393-404Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In human cells, the vimentin (VIM) gene involved in cell and tissue integrity is upregulated in different types of cancer due to antisense transcription of a lncRNA, VIM-AS1, that forms an R loop at the VIM promoter region and transcription start site (TSS) favoring the recruitment of transcription factors. Depletion of the VIM-AS1 or R-loop disruption by RNase H overexpression inactivates VIM expression (Boque-Sastre et al., 2015Boque-Sastre R. Soler M. Oliveira-Mateos C. Portela A. Moutinho C. Sayols S. Villanueva A. Esteller M. Guil S. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation.Proc. Natl. Acad. Sci. USA. 2015; 112: 5785-5790Crossref PubMed Scopus (72) Google Scholar). Similarly, the antisense lncRNA TARID also forms an R loop that regulates gene expression. In this case, TARID is located at the promoter region of the tumor suppressor gene TCF21, and the stress response protein GADD45A binds to this R loop and recruits methylcytosine dioxygenase TET1 leading to local demethylation and gene transcription (Arab et al., 2019Arab K. Karaulanov E. Musheev M. Trnka P. Schäfer A. Grummt I. Niehrs C. GADD45A binds R-loops and recruits TET1 to CpG island promoters.Nat. Genet. 2019; 51: 217-223Crossref PubMed Scopus (23) Google Scholar). In addition to localization at promoters in human genes, R loops are enriched over G-rich terminator elements. These would facilitate RNA Polymerase II (RNAPII) pausing before efficient termination (Figure 1C). The SETX helicase could act on these R loops positioned downstream of gene poly(A) signals (Skourti-Stathaki et al., 2011Skourti-Stathaki K. Proudfoot N.J. Gromak N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination.Mol. Cell. 2011; 42: 794-805Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), where SETX seems to act together with the Tudor-domain protein SMN, which is recruited to the di-methylated CTD of RNAPII (Zhao et al., 2016Zhao D.Y. Gish G. Braunschweig U. Li Y. Ni Z. Schmitges F.W. Zhong G. Liu K. Li W. Moffat J. et al.SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination.Nature. 2016; 529: 48-53Crossref Pub