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R-loops induce repressive chromatin marks over mammalian gene terminators

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 strand1. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes2,3. 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 termination4. 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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Figure 1: The RNAi-dependent H3K9me2 repressive mark is formed over the human β-actin terminator in HeLa cells.
Figure 2: Modulation of R-loop and G9a levels defines the mechanism of H3K9me2 formation on the human β-actin terminator.
Figure 3: Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene.
Figure 4: HP1γ, G9a and R-loops are globally associated with PTT regions.

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Gene Expression Omnibus

Data deposits

Sequencing data for PolIIS2ph ChIP and inputs from BIX RH1-treated and untreated HeLa cells have been deposited in the Gene Expression Omnibus under accession number GSE59878.

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Acknowledgements

This work was supported by a Wellcome Trust Programme grant (091805/Z/10/Z) and a European Research Council Advanced grant (339270-polyloop) to N.J.P., and a Marie Curie Actions grant from EU FP7 (REA grant agreement, 327985) to K.K.-G. We are grateful to R. Nunes Bastos for valuable help with immunofluorescence/imaging analysis and figure formatting. We also thank Q. Sun for helpful discussions and advice. We thank R. J. Crouch for the gift of the GFP-RNase H1 expression plasmid, G. Hannon for the Ago2-knockout mouse cell line and Y. Shinkai for the G9a/Glp double-knockout mouse embryonic stem cells.

Author information

Authors and Affiliations

Authors

Contributions

K.S.-S. performed all the molecular biology and imaging experiments. K.K.-G. performed the bioinformatics analysis. K.S.-S. and N.J.P. designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Nicholas J. Proudfoot.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 H3K9me2 and H3 levels over human β-actin gene.

a, Left, H3K9me2 ChIP on β-actin gene. Right, H3K9me2 ChIP analysis on human centromere 9 (positive control). b, Left, H3 ChIP on β-actin gene. Right, H3 ChIP analysis on human centromere 9. c, Left, H3K9me2 ChIP with or without BIX treatment. Right, H3 ChIP with or without BIX treatment. ChIP values are ±s.d. from three biological repeats. C and D indicate C and D termination probes, respectively. cen, centromere; in, intron; p(A), poly(A); prom, promoter.

Extended Data Figure 2 R-loops and the 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 the 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 ±s.d. from three biological repeats.

Extended Data Figure 3 Cellular localization 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 visualization of R-loops and dsRNA in HeLa cell nuclei. Enlarged boxes (1 and 2) are shown in right panels. b, Whole-cell images showing immunofluorescence of H3K9me2 with dsRNA (J2; top) and R-loops (S9.6; bottom). Enlarged versions (1 and 2) are shown in Fig. 2h.

Extended Data Figure 4 R-loops and RNAi promote the 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 ±s.d. from four biological repeats. c, Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalized to intron 1 signal. d, Left, ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle, normalized H3K9me3 to total H3 levels. Right, ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e, Ago1 ChIP in wild-type (grey bars) and Ago2-knockout (KO) (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f, Left, ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild-type and G9a/Glp double-knockout mouse embryonic stem cells. Right, H3K9me2/H3 ratio on the mouse major satellites in wild-type and G9a/Glp double-knockout cells. Average ChIP and DIP values are ±s.d. from three biological repeats.

Extended Data 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, 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 ±s.d. from three biological repeats.

Extended Data Figure 6 H3K9me2 and H3 levels over mouse β-actin gene in G9a/Glp double-knockout mouse embryonic stem cells and Ago2-knockout MEFs.

a, Top and bottom, H3K9me2 and H3 ChIP performed on mouse β-actin gene in wild-type and G9a/Glp double-knockout embryonic stem cells. H3K9me2 occupancy depends on the presence of G9a/Glp HKMTs. Right, H3K9me2 and H3 ChIP performed on mouse major satellites in wild-type and G9a/Glp double-knockout cells. b, ChIP analyses using H3K9me2 (top) and H3 (bottom) antibodies performed on mouse β-actin gene in wild-type and Ago2-knockout cells. ChIP values are ±s.d. from three biological repeats.

Extended Data Figure 7 R-loop formation and antisense transcription are Ago2- and G9a/GLP-independent.

ac, DIP performed on mouse β-actin gene in wild-type, Ago2-knockout (a) and G9a/Glp double-knockout (c) cells. b, Pol II ChIP in wild-type (grey bars), wild-type overexpressing RNase H1 (black bars), Ago2-knockout (white bars) and Ago2-knockout overexpressing 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 double-knockout cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT–qPCR values are ±s.d. from three biological repeats.

Extended Data Figure 8 HP1γ, G9a and R-loops are globally associated with paused Pol II over PTTs.

a, Genomic annotation of HP1γ based on ChIP-seq peak 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 base-pair coverage in the human genome (genome annotation, pie chart in the middle). Genic regions were 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 summit frequencies 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 = 0.0009337). The Wilcoxon Mann–Whitney test was applied in all cases. d, DRIP-seq profile over the centre of PTT candidate regions. Read frequencies of DRIP sample (black curve) and DRIP RH1 sample (red curve) are plotted in 500-bp bins, both normalized to million mapped reads. e, Box plot showing DRIP-seq2 read density (RPKM) of DRIP sample compared with DRIP RH1 control in PTT candidate regions. P < 2.2 × 10−16 determined by Wilcoxon signed-rank test. f, Box plot of PolIIS2ph pausing index over PTTs (relative to gene bodies) in the BIX RH1 sample (right) and the untreated sample (left). P = 3.398 × 10−16 using the Wilcoxon signed-rank test. g, Box plot displaying the ratio of PolIIS2ph pausing index in the BIX RH1-treated sample compared with the untreated sample in TSS regions (±1 kb, left) and in PTT regions (right). P = 2.468 × 10−15 using the Wilcoxon Mann–Whitney test.

Extended Data Figure 9 ENSA and GEMIN7 share features of R-loop mediated PTT.

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 ± s.d. 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 ±s.d. from four biological repeats. c, DICER ChIP of ENSA and GEMIN7 genes over promoters and termination regions. d, Left, ratio of H3K9me2 ChIP signal versus H3 on GEMIN7 and β-actin genes. Right, 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 ±s.d. from three biological repeats.

Extended Data Figure 10 Model for how R-loops and RNAi-dependent H3K9me2 chromatin mediate pause-type 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 hybridizes 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 before 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.

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Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014). https://doi.org/10.1038/nature13787

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