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R loops regulate promoter-proximal chromatin architecture and cellular differentiation

Nature Structural & Molecular Biology volume 22, pages 999–1007 (2015)Cite this article

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Abstract

Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R loops, RNA-DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin-regulatory complexes, Tip60–p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60–p400 histone acetyltransferase complex binds to nascent transcripts; however, transcription promotes chromatin binding of Tip60–p400 but not PRC2. Interestingly, we observed higher Tip60–p400 and lower PRC2 levels at genes marked by promoter-proximal R loops. Furthermore, disruption of R loops broadly decreased Tip60–p400 occupancy and increased PRC2 occupancy genome wide. In agreement with these alterations, ESCs partially depleted of R loops exhibited impaired differentiation. These results show that R loops act both positively and negatively in modulating the recruitment of key pluripotency regulators.

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Figure 1: Tip60–p400 binds nascent transcripts.
Figure 2: Transcription promotes promoter-proximal association of Tip60–p400.
Figure 3: Promoter-proximal R loops colocalize with Tip60–p400.
Figure 4: R loops promote elevated Tip60 and p400 binding to many target genes.
Figure 5: R loops inhibit chromatin binding by PRC2.
Figure 6: Disruption of R loops impairs ESC differentiation.
Figure 7: Model of R-loop function.

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Acknowledgements

We thank W. Hardy and M. Green (University of Massachusetts Medical School) for the pCAGGS-ires-Hygro expression vector and S. Hainer, L. Ee and K. McCannell for input on the manuscript. This work was supported by US National Institutes of Health (NIH) grant R01HD072122 and American Cancer Society grant RSG-14-220-01 (both to T.G.F.) and NIH grant R01HD080224 (to O.J.R.). T.G.F. was supported as a Pew Scholar in the Biological Sciences and is supported as a Leukemia and Lymphoma Society Scholar. Deep sequencing was performed at the University of Massachusetts Medical School Core facility on a HiSeq2000 supported by NIH grant 1S10RR027052-01.

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Authors and Affiliations

  1. Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Poshen B Chen, Diwash Acharya & Thomas G Fazzio

  2. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Poshen B Chen, Diwash Acharya & Thomas G Fazzio

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Hsiuyi V Chen & Oliver J Rando

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Contributions

P.B.C. performed all experiments and bioinformatics analyses, except as otherwise indicated. H.V.C. prepared libraries for RIP-seq, D.A. generated several of the ESC lines, and T.G.F. performed Suz12 ChIP-seq and analyzed the data. P.B.C., O.J.R. and T.G.F. designed the experiments. P.B.C. and T.G.F. wrote the paper with input from all authors.

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Correspondence to Thomas G Fazzio.

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Integrated supplementary information

Supplementary Figure 1 Characteristics of Tip60–p400–bound transcripts.

a-b, Scatter plots of biological replicate RIP-seq data (Log2 RPKM) for p400 RIPs (a) and Ruvbl1 RIPs (b). Correlation coefficients (R) are noted. c, Higher enrichment of 5’ reads (within the 1st exon and 1st intron) among transcripts from RIP-seq experiments relative to RNA-seq. Shown are cumulative distribution plots indicating a significant increase in the proportion of reads in the first exon and first intron within the p400 RIP-seq libraries relative to RNA-seq. Similar findings were observed for Ruvbl1 RIP-seq. P-value indicating significant enrichment of 5’ proximal reads in RIP-seq data were calculated using a two-sample K-S test. d, Overlap between high confidence Tip60–p400 bound RNAs and genes. The significance of overlap was calculated using a hypergeometric test. e, Tip60–p400-bound transcripts occupy a broad range of expression. Scatter plot of biological replicate RNA-seq data (Log2 RPKM) overlaid with Tip60–p400-bound transcripts (red). f, Gene Ontology (GO) terms significantly overrepresented among Tip60–p400-bound transcripts, plotted as –Log10 (p-value).

Supplementary Figure 2 Acute transcription inhibition inhibits Tip60–p400 binding to chromatin.

a, Western blots of indicated proteins showing unchanged levels of Tip60–p400 subunits p400, Hdac6, Dmap1, Tip60, and Ruvbl1 upon DRB or Triptolide treatment. Actin serves as a loading control. b, Transcript levels of rapidly-degraded transcripts (Nanog, Myc) and slowly-degraded transcripts (Oct4, Gapdh), measured by RT-qPCR. Levels in cells treated with indicated transcription inhibitors are expressed relative to control cells. Error bars, s.d. (n = 3 technical replicates from one of two independent cell cultures for each condition.) c, ChIP-qPCR of the p400 subunit of Tip60–p400 complex with or without transcription inhibitor treatment. IgG serves as a negative control for ChIP. Enrichment levels are expressed for each locus as a fraction of input. Error bars, s.d. (n = 3 technical replicates from one of two independent cell cultures for each condition.) P values were calculated using two tailed students t-tests (*P < 0.05; **P < 0.01).

Supplementary Figure 3 Comparison of DRIP-RNA-seq method to standard DRIP-seq.

a, Published DRIP-seq data from human embryonal carcinoma cells (Ginno, P. et al., Molecular Cell 45:814–825, 2012) were aggregated over TSSs. Control: DRIP from unperturbed sample. RNaseH pre-treatment: samples were digested with RNaseH prior to DRIP, as a negative control for immunoprecipitation with the S9.6 antibody. b, Comparison of DRIP-seq and DRIP-RNA-seq protocol. c, Comparison of sense and antisense reads of DRIP data aggregated near promoters as in (a). d, Dot blots indicating changes in bulk R-loop abundance upon Rnaseh1 overexpression or treatment of cells with DRB or Triptolide. Treatment of purified genomic DNA with RNaseH in vitro (last lane) serves as a control. Significance of differences in (a) and (c) calculated using two-sample K-S tests, as above.

Supplementary Figure 4 Reduction in Tip60–p400 binding to target genes upon Rnaseh1 overexpression.

a-b, Browser tracks of Tip60 or p400 ChIP-seq data at two Tip60–p400 target genes, Rps9 (a) and Rpl8 (b) in control cells or Rnaseh1 overexpressing cells. Gene expression is shown as RNA-seq tracks for each gene. c, Two genes are shown with high (Cdc6) or low (Wipf2) expression levels to illustrate that some lowly expressed genes with R-loops exhibit Rnaseh1-sensitive Tip60–p400 binding, and some highly expressed genes without detectable R-loops are not highly bound by Tip60–p400.

Supplementary Figure 5 ChIP-qPCR validation of changes in factor binding upon Rnaseh1 overexpression.

a-b, ChIP-qPCR measurements of Tip60 (a) or Suz12 (b) association with genes that harbor minimal, modest, or high levels of R-loops, and the effect of R-loop destruction by Rnaseh1 overexpression are shown. Error bars, s.d. (n = 3 technical replicates from one of two independent cell cultures for each line.) P values comparing Rnaseh1 overexpression to controls were calculated using two tailed students t-tests (*P <0.05; **P < 0.01).

Supplementary Figure 6 Effects of Rnaseh1 overexpression on ESC proliferation and self-renewal.

a, Brightfield images or alkaline phosphatase staining of control or Rnaseh1 overexpressing ESCs. b, DNA content of control or Rnaseh1 overexpressing ESCs (X-axis) measured by propidium iodide staining and FACS. One representative curve of three biological replicates is shown for each cell type. c, Quantification of S-phase population for three biological replicate (independent cultures) control and Rnaseh1 overexpressing ESCs. N.S., differences in S-phase population are not statistically significant. d, RT-qPCR quantification of 18S and 28S rRNA levels in control or Rnaseh1 overexpressing ESCs. Data were normalized relative to Gapdh, and rRNA levels in control cells were set to 100%. Shown are technical replicates of one representative biological replicate of two. Error bars = standard deviations. e, Growth curve of three biological replicate (independent cultures) control or Rnaseh1 overexpressing ESCs compared to Ep400 knockdown (KD) ESCs. f, ChIP-qPCR measurements of TSS-proximal and gene body RNA Pol II association with genes that lose Tip60–p400 binding in RNaseH-oex cells show no reduction in RNA Pol II association upon Rnaseh1 overexpression. Error bars, s.d. (n = 3 technical replicates from one of two independent cell cultures for each line.) P values were calculated using two tailed students t-tests (*P < 0.05; **P < 0.01).

Supplementary Figure 7 Enhanced Suz12 binding and H3K27me3 localization upon Rnaseh1 overexpression.

a, Heatmaps, as in Fig. 5, showing promoter-proximal H3K27me3 localization for all genes in control or Rnaseh1 overexpressing ESCs. Heatmaps are sorted by H3K27me3 localization in control cells. b-c, Genes normally targeted by PRC2 show increased Suz12 enrichment in Rnaseh1 overexpressing cells. Browser tracks are depicted as in Fig. 5. Gene expression is shown as RNA-seq tracks for each gene. d, Browser tracks of R-loops and Tip60 and Suz12 association at a locus that switches from Tip60 binding to Suz12 binding upon disruption of R-loops, depicted as in Fig. 5. Gene expression is illustrated with RNA-seq tracks.

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Chen, P., Chen, H., Acharya, D. et al. R loops regulate promoter-proximal chromatin architecture and cellular differentiation. Nat Struct Mol Biol 22, 999–1007 (2015). https://doi.org/10.1038/nsmb.3122

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