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. 2014 Dec 18;516(7531):436-9.
doi: 10.1038/nature13787. Epub 2014 Oct 5.

R-loops induce repressive chromatin marks over mammalian gene terminators

Konstantina Skourti-Stathaki  1 , Kinga Kamieniarz-Gdula  1 , Nicholas J Proudfoot  1
Affiliations

R-loops induce repressive chromatin marks over mammalian gene terminators

Konstantina Skourti-Stathaki et al. Nature. .

Abstract

The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potentially harmful effects on genome integrity owing to the fragility of the displaced DNA coding strand. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes. Furthermore, we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing before efficient termination. Here we reveal an unanticipated link between R-loops and RNA-interference-dependent H3K9me2 formation over pause-site termination regions in mammalian protein-coding genes. We show that R-loops induce antisense transcription over these pause elements, which in turn leads to the generation of double-stranded RNA and the recruitment of DICER, AGO1, AGO2 and the G9a histone lysine methyltransferase. Consequently, an H3K9me2 repressive mark is formed and heterochromatin protein 1γ (HP1γ) is recruited, which reinforces Pol II pausing before efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.

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Figures

ED Figure 1
ED Figure 1. H3K9me2 and H3 levels over human β-actin gene
a. Left panel: H3K9me2 ChIP on β-actin gene. Right panel: H3K9me2 ChIP analysis on human centromere 9 (positive control). b. Left panel: H3 ChIP on β-actin gene. Right panel: H3 ChIP analysis on human centromere 9. c. Left panel: H3K9me2 ChIP +/− BIX treatment. Right panel: H3 ChIP +/− BIX treatment. ChIP values are +/− SD from three biological repeats.
ED Figure 2
ED Figure 2. R-loops and H3K9me2 repressive mark are not specifically enriched over the CoTC terminators of human Cyclin B1 and Akirin 1 genes
a. DIP on endogenous Cyclin B1 and Akirin 1 genes. No detection of R-loops was observed over their CoTC terminators. Human β-actin gene was used as a positive control. For Cyclin B1 and Akirin 1 genes, 3′ end 1 and 3′ end 2 amplicons amplify two different regions within the CoTC terminator of each gene. 3′ end 1 and 3′ end 2 amplicons for β-actin gene amplify the 5′ pause and pause amplicons, respectively. b. Ratio of H3K9me2 signal over the 3′ ends versus intron 3 signal in Cyclin B1, Akirin 1 and β-actin human genes. DIP and ChIP values are +/− SD from three biological repeats.
ED Figure 3
ED Figure 3. Cellular localisation of R-loops, dsRNA and H3K9me2
a. Immunofluorescence imaging of dsRNA (J2 antibody) and R-loops (S9.6 antibody), using paraformaldehyde (PFA) and methanol (MeOH) as fixing reagents. Fixation with methanol allowed visualisation of R-loops and dsRNA in HeLa cell nuclei. Enlarged boxes (1 and 2) shown in right panels. b. Whole cell images showing immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Enlarged versions (1 and 2) are shown in Fig. 2h.
ED Figure 4
ED Figure 4. R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator
a. DIP performed on mouse β-actin gene in MEFs. b. RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c. Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e. Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f. Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.
ED Figure 5
ED Figure 5. H3K9me2, H3K9me3 and H3 levels over the endogenous mouse β-actin gene
a. H3K9me2 and H3K9me3 ChIP on mouse β-actin gene in MEF cells. Right panel: H3K9me2 and H3K9me3 ChIP on mouse major satellites (positive control). b. Total H3 ChIP on mouse β-actin gene. Major satellites were used as a positive control. ChIP values are +/− SD from three biological repeats.
ED Figure 6
ED Figure 6. H3K9me2 and H3 levels over mouse β-actin gene in G9a/GLP double KO mouse ES cells and Ago2 KO MEFs
a. Top and bottom panels: H3K9me2 and H3 ChIP performed on mouse β-actin gene in wild type and G9a/GLP double KO ES cells. H3K9me2 occupancy depends on presence of G9a/GLP HKMTs. Right panels: H3K9me2 and H3 ChIP performed on mouse major satellites in wild type and G9a/GLP KO cells. b. ChIP analyses using H3K9me2 (top panel) and H3 (bottom panel) antibodies performed on mouse β-actin gene in wild type and Ago2 KO cells. ChIP values are +/− SD from three biological repeats.
ED Figure 7
ED Figure 7. R-loop formation and antisense transcription are Ago2 and G9a/GLP-independent
a-c DIP performed on mouse β-actin gene in wild type, Ago2 KO (a) and G9a/GLP KO (c) cells. b. Pol II ChIP in wild type (grey bars), wild type over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Hatched box quantifies Pol II read-through transcription versus promoter signal. d. RT-qPCR analysis of total RNA from wild type and G9a/GLP KO cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT-qPCR values are +/− SD from three biological repeats.
ED Figure 8
ED Figure 8. HP1γ, G9a and R-loops are globally associated with paused Pol II over pause-type termination regions (PTTs)
a. Genomic annotation of HP1γ based on ChIP-seq peaks summit localization (HP1γ annotation, pie chart on the left) and the fold enrichment of HP1γ over the indicated genomic regions (table on the right) as compared to their bp coverage in the human genome (genome annotation, pie chart in the middle). Genic regions have been defined by RefSeq gene coordinates (hg19). Promoter regions were defined as regions 1 kb upstream of RefSeq gene TSS excluding intervals overlapping with any genic regions. Termination regions were defined as regions 5 kb downstream of RefSeq genes excluding intervals overlapping with any genic region or promoter. b. HP1γ ChIP-seq enrichment profile in 10 kb regions surrounding the TSS (left graph) and PAS (right graph). HP1γ peaks summits frequency are plotted in 500 bp bins.c. Box plot showing the average log2 (G9a/input) ChIP-chip signal distribution in PTT candidate regions (right box), randomly sampled regions of the same size and number as PTT candidate regions (random regions, left box), and in HP1γ peaks outside of PTT candidate regions (non-PTT HP1γ peaks, middle box). In all box plots the horizontal line in the box shows the median, the lower and upper limits of the box show respectively the first and third quartile, and the whiskers extend to the non-outlier extreme data points. The log2 (G9a/input) signal is significantly higher in the PTT candidate regions compared to random regions (p=0.0001067) as well as compared to non-PTT HP1γ peaks (p=0.02213). The log2 (G9a/input) signal is also significantly higher in non-PTT HP1γ peaks compared to random regions (p=0.0.0009337). The Wilcoxon-Mann-Whitney test has been applied in all cases. d. DRIP-seq profile over centre of PTT candidate regions. Read frequencies of DNA:RNA-IP sample (DRIP, black curve) are plotted and DNA:RNA-IP sample treated with RNase H1 (DRIP RH1, red curve) in 500 bp bins, both normalized to million mapped reads. e. Box plot showing DRIP-seq read density (RPKM) of DNA:RNA-IP (DRIP) sample compared with DRIP sample treated with RNase H1 (DRIP RH1 control) in PTT candidate regions. P<2.2e-16 determined by Wilcoxon signed-rank test. Horizontal line in the box shows median, box lower and upper limit show first and third quartile, whiskers extend to non-outlier extreme data points. f. Box plot of PolIIS2ph pausing index over PTTs (relative to gene bodies) in the BIX RH1 sample (right) with the untreated sample (left). P=3.398e-16 using Wilcoxon signed-rank test. g. Box plot displaying ratio of PolIIS2ph pausing index in the BIX RH1 to untreated sample in TSS regions (+/−1 kb, left) and in PTT regions (right). P=2.468e−15 using Wilcoxon-Mann-Whitney test.
ED Figure 9
ED Figure 9. Ensa and Gemin7 share features of R-loop mediated pause-type termination
a. DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b. RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c. Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g. HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.
ED Figure 10
ED Figure 10. Model for how R-loops and RNAi-dependent H3K9me2 chromatin mediate pause-dependent transcriptional termination in mammalian genes
Mammalian genes possessing pause elements downstream of their PAS form R-loops in termination regions. This facilitates generation of an antisense transcript that hybridises with the sense transcript to form dsRNA. This triggers recruitment of the RNAi factors, Dicer, Ago1 and Ago2. G9a/GLP HKMTs and HP1γ are then recruited forming and maintaining H3K9me2 repressive marks. R-loops and H3K9me2 facilitate Pol II pausing prior to termination. DNA is shown as grey lines and RNA as a red line. Points of contact between the DNA strand and nascent RNA indicates R-loop formation, whereas points of contact between sense and antisense RNA indicate dsRNA formation. Pol II is shown as a blue icon with arrow indicating transcription direction. Nucleosomes are shown in green except over H3K9me2 region where they are coloured red.
Figure 1
Figure 1. RNAi-dependent H3K9me2 repressive mark formed over human β-actin terminator in HeLa cells
a. RT-qPCR of β-actin antisense transcription. RT with region-specific forward primers. b. Sense and antisense transcripts levels by RT-qPCR from J2 immuno-selected dsRNA. Samples either untreated (grey bars), treated with V1 RNase (black bars) or S1 nuclease (white bars). All RT-qPCR values are average +/− SD from three to four biological repeats. c-e, g. ChIP analysis using Dicer, Ago1, G9a and HP1γ antibodies respectively. f. Ratio of H3K9me2 ChIP versus H3 on β-actin gene and centromere 9 (right panel). ChIP values +/− SD from three biological repeats.
Figure 2
Figure 2. Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator
a. DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b. RT-qPCR with/without RNase H1 over-expression. c-e. ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f. H3K9me2 versus H3 ChIP values, +/− BIX treatment. g. DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats.h. Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b. Cell numbers with >2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).
Figure 3
Figure 3. Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene
a,b. ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c. Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d. Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b. All ChIP values +/− SD from three to four biological repeats. e. Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.
Figure 4
Figure 4. HP1γ, G9a and R-loops are globally associated with PTT regions
a. Diagram of pause-type termination (PTT) candidate regions. PTT candidate regions were defined as genomic intervals delineated by ChIP-seq peaks of PolIIS2ph (ENCODE) overlapping with ChIP-seq peaks of HP1γ within termination regions (5 kb downstream of human RefSeq genes, not overlapping with any downstream gene or promoter). b. Bar graph displaying the observed (2064) and expected based on random sampling (115) overlap of HP1γ terminator peaks with PolIIS2ph peaks. c. G9a ChIP-chip profile over PTT candidate regions (black curve) and non-PTT associated HP1γ peaks (other HP1γ peaks, red curve). d. Br-UTP NRO analysis −/+ BIX treatment with RNase H1 over-expression on Ensa, Gemin7 and β-actin genes. -Fold of enrichment of read-through transcripts over gene 3′ end calculated relative to intronic signals (set as 1). Values +/− SD from three biological repeats. e. PolIIS2ph ChIP-seq enrichment profiles for untreated (blue curve) and BIX plus RNAse H1 (RH1) overexpression (red curve) in 15 kb regions over centre of TSS (left graph) and PAS (right graph).
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