Abstract
RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3′ end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3′-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m6A writer complex, and m6A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.
Highlights
RNA-mediated chromatin silencing is central to genome regulation in many organisms
The COOLAIR R-loop at the 3′-end of FLC is stabilized by the homeodomain protein AtNDX, which inhibits further antisense transcription[11]
The R-loop corresponds in length to the nascent short proximal COOLAIR (~626 nucleotides) (Fig. 1a), whose formation is promoted by the RNA-binding proteins FCA and FPA and the canonical 3′-end processing factors FY, CstF64, and CstF777,18
Summary
RNA-mediated chromatin silencing is central to genome regulation in many organisms. how nascent non-coding transcripts regulate chromatin is poorly understood. Stabilization of an antisense-induced R-loop at the 3′ end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3′-end processing factors, to polyadenylate the nascent antisense transcript This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me[1]. Genetic screens have identified RNA-binding proteins (FCA, FPA, and FLK), conserved 3′-end processing factors (FY/WDR33, CstF64, CstF77), the PRP8 splicing factor, and FLD a histone demethylase homolog, as components necessary for this transcriptional silencing[3,4,5,6] These co-transcriptional regulators promote proximal polyadenylation of COOLAIR7–9, which leads to the recruitment of a set of physically interacting chromatin modifiers FLD/LD/SDG2610. We describe a mechanism in which modulation of R-loop stability by co-transcriptional RNA processing involving m6A modification can trigger chromatin silencing
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