R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes

Skourti-Stathaki, Konstantina; Triglia, Elena Torlai; Warburton, Marie; Voigt, Philipp; Bird, Adrian; Pombo, Ana

doi:10.1016/j.molcel.2018.12.016

Cited by 83 publications

(76 citation statements)

References 72 publications

(134 reference statements)

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“…Our results suggest a new mechanism for targeting PcG function through R-loop formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.…”

supporting

confidence: 57%

“…One way for RNA to interact with the genome is by the formation of R-loops, three-stranded nucleic acid structures formed when an RNA hybridizes to a complementary DNA strand, thereby displacing the second DNA strand 5 . The formation of R-loops over genes with low to moderate expression is associated with increased PcG binding and H3K27 trimethylation (H3K27me3) in human cells 25 and R-loops have been implicated in promoting PcG recruitment in mammalian cells 6 , although other evidence suggests they antagonize recruitment 26 . We hypothesized that R-loop formation could biochemically link RNA to silencing through PREs and tested this idea in the Drosophila system.…”

Section: Main Textmentioning

confidence: 99%

“…The connection between RNA and PRC2 has been recognized for some time, in species from plants to humans 16-18,35 , but mechanisms beyond RNA binding by PRC2 have not previously been described. Our discovery of PRC2-mediated RNA strand invasion, and R-loop formation at PREs, suggests a mechanism to connect RNA to PcG targeting and function, and the formation and recognition 6 of non-canonical nucleic acid structures to epigenetics (Extended Data 10).…”

Section: Main Textmentioning

confidence: 99%

See 2 more Smart Citations

RNA strand invasion activity of the Polycomb complex PRC2

Alecki

Chiwara

Sanz

et al. 2019

Preprint

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Epigenetic regulation is conveyed through information encoded by specific chromatin 20 features. Non-canonical nucleic acid structures could in principle also convey biological 21 information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) 22 proteins form memory of transient transcriptional repression events that is necessary for 23 development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb 24 Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, 25 active, or unengaged states 1,2 . How PcG activities are targeted to PREs to maintain repressed 26 states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, 27PcG protein complexes modify chromatin to maintain gene repression 1,3,4 . However, PcG 28 proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG 29 function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA 30 hybridizes to its complementary DNA and displaces the other DNA strand 5 , form at many PREs 31 in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the 32 PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand 33 invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-34 loops. Our results suggest a new mechanism for targeting PcG function through R-loop 35 formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation 36 and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.37 38 39 40 41 Main Text: 42 During Drosophila embryogenesis, transiently expressed transcription factors activate 43 homeotic (Hox) genes in certain regions of the embryo and repress them in others to dictate the 44 future body plan 7 . Polycomb Group (PcG) proteins form a memory of these early cues by 45 maintaining patterns of Hox gene repression for the rest of development 1,7,8 . This paradigm for 46 transcriptional memory is believed to be used by the PcG at many genes in Drosophila, and to 47 underlie the conserved and essential functions of PcG proteins in cell differentiation and 48 development from plants to mammals 9,10 . Polycomb Response Elements (PREs) are DNA 49 elements that can recruit PcG proteins, but they also recapitulate the memory function of the PcG-50 when combined with early acting, region-specific enhancers in transgenes, they maintain transgene 51 repression in a PcG-dependent manner only in regions where the early enhancer was not active 52 1,11,12 . PREs contain a high density of binding sites for transcription factors that can recruit PcG 53 proteins through physical interactions 12 . However, the widespread expression, binding pattern, 54 and properties of factors that bind PREs cannot explain how PREs can exist in alternate, 55 transcription-history dependent states to maintain restricted patterns of gene expression, or how 56

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supporting

confidence: 57%

Section: Main Textmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

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RNA strand invasion activity of the Polycomb complex PRC2

Alecki

Chiwara

Sanz

et al. 2019

Preprint

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“…This includes links to PcG regulation in mammalian cells. The formation of R-loops over genes with low to moderate expression is associated with increased PcG binding and H3K27 trimethylation (H3K27me3) in human cells 24 and R-loops have recently been implicated in promoting PRC1 and PRC2 recruitment in mammalian cells 25 , although other evidence suggests they antagonize recruitment of PRC2 26 . We hypothesized that R-loop formation could biochemically link RNA to PcG-mediated silencing through PREs and tested this idea in the Drosophila system.…”

mentioning

confidence: 99%

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

et al. 2020

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Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

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“…R-loops have been described from bacteria to plants to mammals (Santos-Pereira and Aguilera, 2015) and represent a prevalent class of alternative DNA structures (Chedin, 2016). Studies over the last decade have implicated R-loop formation in a variety of physiological processes such as transcription termination (Proudfoot, 2016;Sanz et al, 2016;Skourti-Stathaki et al, 2011), chromatin patterning and gene expression control (Chedin, 2016;Niehrs and Luke, 2020;Sanz et al, 2016;Skourti-Stathaki et al, 2019;Tan-Wong et al, 2019), and class switch recombination (Yu et al, 2003;Yu and Lieber, 2019). By contrast, so-called aberrant R-loops formed under a variety of pathological conditions have been extensively linked to phenomena of genome instability (Crossley et al, 2019;Garcia-Muse and Aguilera, 2019;Hamperl and Cimprich, 2014).…”

Section: Introductionmentioning

confidence: 99%

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

Castillo-Guzman

Hartono

Sanz

et al. 2020

Preprint

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Efficient co-transcriptional splicing is thought to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. Inhibition of the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells caused widespread intron retention and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these events, arguing that unspliced introns do not cause excessive R-loops. R-loop gains were instead driven by readthrough transcription resulting from loss of transcription termination over a subset of stress-response genes, defining a new class of aberrant "downstream of genes" (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss resulting from accumulation of promoter-proximal paused RNA polymerases and defective elongation. Thus, SF3B1-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in the global R-loop landscape. HIGHLIGHTS • Intron retention caused by SF3B1 inhibition does not lead to excessive R-loops • A subset of genes shows readthrough transcription and accompanying R-loop gains • SF3B1 inhibition causes broad reduction in nascent transcription and R-loop loss • R-loop gains and DNA damage are temporally and spatially uncoupled RNAPII RNA +PladB PAS zZz Promoter pausing Elongation R-loops downstream of gene PAS zZz R-loops Txn read through DoG R-loops Promoter pausing Elongation No R-loop PAS zZz

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R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes

Cited by 83 publications

References 72 publications

RNA strand invasion activity of the Polycomb complex PRC2

RNA strand invasion activity of the Polycomb complex PRC2

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

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