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.
Highlights
Around forty human diseases are associated with expanded repeat sequences [1]
Friedreich ataxia and Fragile X syndrome are among 40 human diseases associated with expansion of repeated sequences
It is proposed that formation of unusual DNA structures (RNA/DNA hybrids, or R-loops) over repeat regions may play a role, but their molecular function has not been investigated in vivo
Summary
Around forty human diseases are associated with expanded repeat sequences [1]. Friedreich ataxia (FRDA) is the most frequent autosomal recessive ataxia (2–4 cases/100,000), caused by a GAA expansion in the first intron of the frataxin (FXN) gene, which encodes a mitochondrial protein involved in iron-sulfur cluster biogenesis [2,3]. Several mechanisms mediating FXN transcriptional silencing have been proposed, including the formation of unusual DNA structures (triplex DNA and RNA/DNA hybrids) and repressive heterochromatin over expanded repeats [5,6,7,8,9,10]. We recently showed that R-loops formed over the G-rich pause sites downstream of the polyA signal in human genes are essential for the process of transcriptional termination of RNA Pol II [16]. RNA/DNA hybrids are induced at GAA repeats following in vitro transcription and in bacteria [17,18]. R-loops formed on plasmids containing CTG/CAG repeats in E.coli and mini-gene constructs in human cells promoted repeat instability, pointing towards their role in disease pathology [19,20]. The direct involvement of R-loops on endogenous expanded alleles in the pathology of FRDA has not yet been investigated in vivo
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