Nascent Connections: R-Loops and Chromatin Patterning

Chédin, Frédéric

doi:10.1016/j.tig.2016.10.002

Cited by 185 publications

(181 citation statements)

References 110 publications

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“…Next we increased the GC-content in the R-loop sequences to test whether this would determine high affinity binding, since R-loops are predominantly formed at GC-rich genomic regions [35]. We have shown no binding or weak binding to sequences in the range from 0 to 25% CG content, identified strong binding to sequence with 52% CG content and weak binding to a sequence with 75% GC content (22GC75).…”

Section: Resultsmentioning

confidence: 99%

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

König¹,

Schubert²,

Längst³

2017

PLoS ONE

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The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv) of S9.6 showed high affinity (0.6 nM) for DNA—RNA and also a high affinity (2.7 nM) for RNA—RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

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Section: Resultsmentioning

confidence: 99%

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

König¹,

Schubert²,

Längst³

2017

PLoS ONE

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“…Excessive R-loop formation may also result from defects in RNA export (Huertas and Aguilera, 2003) or pre-mRNA splicing (Li and Manley, 2005), indicating that the commitment of nascent RNA to co-transcriptional RNA-processing pathways in a timely manner helps prevent excessive R-loop formation in normal cells (Bonnet et al, 2017). Despite these advances, many pressing questions remain in understanding R-loop biology with respect to its formation and dynamics in relationship to regulated gene expression, especially at the genome scale (Chédin, 2016). …”

Section: Introductionmentioning

confidence: 99%

“…In recent years, this affinity probe has been coupled with deep sequencing to detect R-loops genome-wide in yeast (Chan et al, 2014; El Hage et al, 2014; Wahba et al, 2016), plant (Xu et al, 2017), and mammals (Ginno et al, 2012, 2013; Nadel et al, 2015; Sanz et al, 2016; Stork et al, 2016). The majority of these methods use the S9.6 antibody to capture RNA/DNA hybrids from restriction digested genomic DNA followed by deep sequencing of captured DNA (DNA:RNA immunoprecipitation [DRIP] sequencing [DRIP-seq] and its derivatives) or RNA (DNA:RNA immunoprecipitation followed by cDNA conversion [DRIPc]-seq and its derivatives) (Chédin, 2016). Because of limited resolution with restriction digestion, additional efforts have also been made to use sonication to increase R-loop mapping resolution (Halász et al, 2017; Nadel et al, 2015); however, such treatment may destroy some fragile R-loops in the absence of fixation.…”

Section: Introductionmentioning

confidence: 99%

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

et al. 2017

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SUMMARY R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

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“…R-loops are widely mapped genome-wide by exploiting the strong affinity of the S9.6 antibody for DNA:RNA hybrids [5,6]. Recent maps have shown that R-loops can occupy between 5% and 10% of the genome in yeast, vertebrates and Arabidopsis thaliana [7–9].…”

Section: Introductionmentioning

confidence: 99%

“…However, a mounting body of evidence also suggests that R-loops play physiological roles in transcriptional control and chromatin patterning (reviewed in Refs. [1,2,5]). What distinguishes toxic and beneficial R-loops is still unclear.…”

Section: Introductionmentioning

confidence: 99%

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

Hartono

Malapert

Legros

et al. 2018

Journal of Molecular Biology

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127

117

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R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

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Nascent Connections: R-Loops and Chromatin Patterning

Cited by 185 publications

References 110 publications

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

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