Abstract
SummaryR-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.
Highlights
During transcription, nascent RNA can hybridize with the DNA template strand, leaving the non-template DNA strand single stranded
R-Loops Form over Polycomb group (PcG)-Repressed Genes To investigate whether R-loops play a role in the PcG-mediated transcriptional silencing, we measured their presence over a panel of PcG target genes using DNA-RNA immunoprecipitation (DIP or DRIP) analysis (Skourti-Stathaki et al, 2011; Ginno et al, 2012, 2013; Skourti-Stathaki et al, 2014; Sanz et al, 2016) in mouse embryonic stem cells (mESCs)
We chose five previously characterized genes, namely Msx1, Math1, Nkx2.2, Nkx2.9, and Gata4. These genes have well-annotated CpG island promoters, are GC-rich throughout their promoters and coding regions, and in mESCs are co-occupied by Polycomb repressive complex 1 (PRC1), PcG-repressed genes. Constitutive EZH2 (PRC2), and poised serine5 phosphorylation (Ser5P) polymerase II (Pol II) (Stock et al, 2007; Brookes et al, 2012; Ferrai et al, 2017)
Summary
Nascent RNA can hybridize with the DNA template strand, leaving the non-template DNA strand single stranded These structures are called R-loops, and their persistent formation can cause deleterious effects on genome integrity, possibly due to the unpaired single-stranded DNA (ssDNA) (Aguilera and Garcıa-Muse, 2012; Hamperl and Cimprich, 2014; Skourti-Stathaki and Proudfoot, 2014). Even though R-loops have the potential to form over a large proportion of the genome, they are not a simple consequence of transcription. R-loops can act as transcriptional activators, but they can induce transcriptional repression in different cell types and via various mechanisms (Nakama et al, 2012; CastellanoPozo et al, 2013; Powell et al, 2013; Sun et al, 2013; Skourti-Stathaki et al, 2014). This ‘‘dual’’ function of R-loops in activation or repression strongly suggests that R-loop formation can have different roles and mechanisms in different contexts
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