R-loops positioned near promoters of active genes maintain CGIs in an unmethylated state ( Ginno et al., 2012), likely by reducing the affinity of DNMT1 binding to DNA ( Grunseich et al., 2018), or recruiting ten-eleven translocation (TET) methylcytosine dioxygenases ( Arab et al., 2019). More than 70% of all human gene promoters contain stretches of CpG dinucleotides, termed CpG islands (CGIs), whose transcriptional activity is repressed by CpG methylation ( Greenberg and Bourc’his, 2019 Weber et al., 2007). The activity of DNA methyltransferase (DNMT) enzymes makes 5mC widespread across the mammalian genome where it plays major roles in imprinting, retrotransposon silencing, and gene expression ( Greenberg and Bourc’his, 2019). DNA methylation, namely, 5-methylcytosine (5mC), results from the covalent addition of a methyl group to the carbon 5 of a C attached to a G through a phosphodiester bond (CpG) ( Karpf, 2013). R-loops formed over G-rich terminator elements promote histone H3 lysine-9 dimethylation, a repressive mark that reinforces RNA polymerase II pausing during transcription termination ( Skourti-Stathaki and Proudfoot, 2014 Chédin, 2016 Skourti-Stathaki et al., 2014).īesides affecting histone modifications, R-loops also act as barriers against DNA methylation spreading into active genes ( Ginno et al., 2013 Ginno et al., 2012). Promoter-proximal R-loops enhance the recruitment of the Tip60–p400 histone acetyltransferase complex and inhibit the binding of polycomb-repressive complex 2 and histone H3 lysine-27 methylation ( Chen et al., 2015). R-loops can also drive chromatin modifications. Moreover, chromatin and DNA features such as histone modifications, DNA-supercoiling, and G-quadruplex structures also affect R-loop establishment ( García-Muse and Aguilera, 2019). An asymmetrical distribution of guanines (G) and cytosines (C) nucleotides in the DNA duplex also influences R-loop propensity, with an excess of Cs in the template DNA strand (positive G:C skew) favoring R-loop formation ( Ginno et al., 2013). The presence of introns, for instance, prevents unscheduled R-loop formation at active genes ( Bonnet et al., 2017). Intrinsic features of the transcribed DNA also influence its propensity to form R-loops. For instance, we previously reported that the DEAD-box helicase 23 (DDX23) resolves R-loops formed during transcription elongation to regulate gene expression programs and prevent transcription-dependent DNA damage ( Sridhara et al., 2017). Several helicases unwind R-loops at different stages of the transcription cycle and in distinct physiological contexts ( García-Muse and Aguilera, 2019). DNA and RNA helicases unwind the hybrid and restore the double-stranded DNA (dsDNA) structure. The ribonuclease H enzymes RNase H1 and RNase H2 degrade R-loops by digesting the RNA strand of the DNA:RNA hybrid. R-loop formation is restricted by RNA-binding proteins and topoisomerase 1, whereas R-loops are removed by ribonucleases and helicases (reviewed in García-Muse and Aguilera, 2019). To preserve genome integrity, cells possess diverse mechanisms to prevent the formation of R-loops or resolve them. However, nonscheduled or persistent R-loops constitute an important source of DNA damage, namely, DNA double-strand breaks (DSBs) ( García-Muse and Aguilera, 2019). These triple-stranded structures, called R-loops, are physiologically relevant intermediates of several processes, such as immunoglobulin class-switch recombination and gene expression ( García-Muse and Aguilera, 2019). Editor's evaluationĭuring transcription, the nascent RNA molecule can hybridize with the template DNA and form a DNA:RNA hybrid and a displaced DNA strand. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming.
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