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A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element

Nature Genetics volume 46, pages 973981 (2014) | Download Citation

Abstract

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.

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References

  1. 1.

    & Transcriptional regulation by Polycomb group proteins. Nat. Struct. Mol. Biol. 20, 1147–1155 (2013).

  2. 2.

    & What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory. Nat. Rev. Mol. Cell Biol. 15, 340–356 (2014).

  3. 3.

    & Polycomb response elements and targeting of Polycomb group proteins in Drosophila. Curr. Opin. Genet. Dev. 16, 476–484 (2006).

  4. 4.

    & Non-coding RNAs in polycomb/trithorax regulation. RNA Biol. 6, 129–137 (2009).

  5. 5.

    , & RNA templating the epigenome: long noncoding RNAs as molecular scaffolds. Epigenetics 6, 539–543 (2011).

  6. 6.

    Noncoding RNA and Polycomb recruitment. RNA 19, 429–442 (2013).

  7. 7.

    et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007).

  8. 8.

    , , , & Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750–756 (2008).

  9. 9.

    et al. Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2. Mol. Cell 38, 675–688 (2010).

  10. 10.

    et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).

  11. 11.

    et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120–124 (2011).

  12. 12.

    et al. Intronic RNAs mediate EZH2 regulation of epigenetic targets. Nat. Struct. Mol. Biol. 19, 664–670 (2012).

  13. 13.

    , , & Promiscuous RNA binding by Polycomb repressive complex 2. Nat. Struct. Mol. Biol. 20, 1250–1257 (2013).

  14. 14.

    , , & Regulatory interactions between RNA and Polycomb repressive complex 2. Mol. Cell 10.1016/j.molcel.2014.05.009 (29 May 2014).

  15. 15.

    , & Allocation of the thoracic imaginal primordia in the Drosophila embryo. Development 117, 597–608 (1993).

  16. 16.

    , & Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5, 2481–2495 (1991).

  17. 17.

    , , & Alternative requirements for Vestigial, Scalloped, and Dmef2 during muscle differentiation in Drosophila melanogaster. Mol. Biol. Cell 20, 256–269 (2009).

  18. 18.

    et al. Control of apterous by vestigial drives indirect flight muscle development in Drosophila. Dev. Biol. 260, 391–403 (2003).

  19. 19.

    , & Vestigial expression in the Drosophila embryonic central nervous system. Dev. Dyn. 237, 2483–2489 (2008).

  20. 20.

    et al. Polycomb preferentially targets stalled promoters of coding and noncoding transcripts. Genome Res. 21, 216–226 (2011).

  21. 21.

    et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471, 480–485 (2011).

  22. 22.

    , , & Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs. Nature 438, 234–237 (2005).

  23. 23.

    et al. Enhancer-PRE communication contributes to the expansion of gene expression domains in proliferating primordia. Development 138, 3125–3134 (2011).

  24. 24.

    et al. Dynamic regulation by polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila. Dev. Cell 15, 877–889 (2008).

  25. 25.

    et al. Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat. Genet. 38, 700–705 (2006).

  26. 26.

    , , & Quantitative analysis of polycomb response elements (PREs) at identical genomic locations distinguishes contributions of PRE sequence and genomic environment. Epigenetics Chromatin 4, 4 (2011).

  27. 27.

    The embryonic development of larval muscles in Drosophila. Development 110, 791–804 (1990).

  28. 28.

    , , & The role of Polycomb-group response elements in regulation of engrailed transcription in Drosophila. Development 135, 669–676 (2008).

  29. 29.

    et al. Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila. Cell 144, 214–226 (2011).

  30. 30.

    , , , & PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1258–1264 (2013).

  31. 31.

    et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res. 21, 182–192 (2011).

  32. 32.

    , & Integrated analysis of the genome and the transcriptome by FANTOM. Brief. Bioinform. 5, 249–258 (2004).

  33. 33.

    et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

  34. 34.

    et al. Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells. Cell 139, 1290–1302 (2009).

  35. 35.

    , & Flowering time control: another window to the connection between antisense RNA and chromatin. Trends Genet. 28, 445–453 (2012).

  36. 36.

    , , & Transcription cofactor Vgl-2 is required for skeletal muscle differentiation. Genesis 39, 273–279 (2004).

  37. 37.

    & Gene regulation by antisense transcription. Nat. Rev. Genet. 14, 880–893 (2013).

  38. 38.

    , , & Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary. Nature 368, 299–305 (1994).

  39. 39.

    et al. Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382, 133–138 (1996).

  40. 40.

    et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  41. 41.

    , & FISH in whole-mount Drosophila embryos. RNA: activation of a transcriptional locus, DNA: gene architecture and expression. Bioimaging 4, 107–120 (1996).

  42. 42.

    , & Cell cycling and patterned cell proliferation in the wing primordium of Drosophila. Proc. Natl. Acad. Sci. USA 93, 640–645 (1996).

  43. 43.

    , , & Model for the regulation of size in the wing imaginal disc of Drosophila. Mech. Dev. 124, 318–326 (2007).

  44. 44.

    et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156 (2007).

  45. 45.

    , & A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila. PLoS ONE 7, e42102 (2012).

  46. 46.

    et al. Elements of the polycomb repressor SU(Z)12 needed for histone H3-K27 methylation, the interface with E(Z), and in vivo function. Mol. Cell. Biol. 33, 4844–4856 (2013).

  47. 47.

    et al. Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin. Nucleic Acids Res. 41, 5235–5250 (2013).

  48. 48.

    , & RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat. Protoc. 1, 302–307 (2006).

  49. 49.

    , , , & Binding of trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis. EMBO J. 17, 5141–5150 (1998).

  50. 50.

    & The Drosophila Enhancer of zeste gene encodes a chromosomal protein: examination of wild-type and mutant protein distribution. Development 122, 4073–4083 (1996).

  51. 51.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  52. 52.

    MULTOVL: fast multiple overlaps of genomic regions. Bioinformatics 28, 3318–3319 (2012).

  53. 53.

    et al. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat. Methods 5, 829–834 (2008).

  54. 54.

    et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

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Acknowledgements

We thank S. Gasser for discussions and for critical reading of the manuscript. We thank M. Rehmsmeier and members of our laboratories for discussions, P. Pasierbek for advice and training on imaging, P.A. Steffen (IMBA) for the GFP and E(z)GFP constructs, C. Ehrhardt and E. Dworschak for technical assistance, B. Dickson (Janelia Farm) for the enGAL4 driver line, J.M. Dura (Institute of Human Genetics, Montpelier, France) for the daGAL4 driver line, I. Tamir (CSF Vienna) for the bioinformatics analysis of ChIP-seq data and sharing the 'Fuge' algorithm, F. Bantignies (Institute of Human Genetics, Montpelier, France) for advice on three-dimensional DNA FISH, R. Jones (Dedman College, Southern Methodist University) for providing antibody to E(Z) and the Vienna Campus Support Facility (CSF) for library preparation, deep sequencing and the purification of Drosophila PRC2. This work was funded by the Austrian Academy of Sciences, by European Community grants European Union Framework Programme 6 Network of Excellence 'The Epigenome' (to L.R.) and the European Union Framework Programme 7 Network of Excellence 'Epigenesys' (to L.R.), and by an FWF Austrian Science Fund grant (P21525-B20 to L.R.).

Author information

Author notes

    • Veronika A Herzog
    •  & Adelheid Lempradl

    These authors contributed equally to this work.

Affiliations

  1. IMBA (Institute of Molecular Biotechnology), Vienna, Austria.

    • Veronika A Herzog
    • , Adelheid Lempradl
    • , Johanna Trupke
    • , Helena Okulski
    • , Christina Altmutter
    • , Frank Ruge
    • , Andrew Dimond
    • , Hasene Basak Senergin
    •  & Leonie Ringrose
  2. Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.

    • Adelheid Lempradl
    • , Marius Ruf
    •  & Andrew Pospisilik
  3. CeMM (Research Center for Molecular Medicine), Vienna, Austria.

    • Bernd Boidol
    •  & Stefan Kubicek
  4. IMP (Institute of Molecular Pathology), Vienna, Austria.

    • Gerald Schmauss
    •  & Karin Aumayr
  5. The Babraham Institute, Babraham Research Campus, Cambridge, UK.

    • Andrew Dimond
  6. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.

    • Marcus L Vargas
    •  & Jeffrey A Simon

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Contributions

A.L. and L.R. initiated the project. L.R., V.A.H. and A.L. designed the experiments. J.T. performed bioinformatics analysis of genome-wide data sets. G.S. and K.A. performed automated image analysis of three-dimensional DNA FISH. V.A.H., A.L., H.O., C.A., F.R., B.B., A.D., M.R., H.B.S. and L.R. conducted the experiments and analyzed the data. S.K. supervised B.B. A.P. supervised M.R. M.L.V. and J.A.S. provided purified Drosophila PRC2 and polynucleosome substrates. L.R. prepared the manuscript with input from V.A.H., J.T., A.L., B.B. and H.O.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Leonie Ringrose.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Tables 1 and 3.

Excel files

  1. 1.

    PcG-bound regions in fly and mouse that show convergent or divergent transcripts.

    Genomic coordinates of PcG-bound regions with the identity of the closest gene. See the Online Methods and legend in the table for details. (a) mouse_CAGE. Mouse ES cell PcG peaks from ref. 34 that overlap with convergent or divergent CAGE tags from the FANTOM3 CAGE dataset32. In mouse, the CAGE data provide tags from multiple developmental stages and tissues, and the majority of the PcG-bound peaks have multiple tags. Individual tags are not listed in the table, and the coordinates of these are available on request. (b) Fly_modENCODE_MACE. Fly PcG peaks from ref. 21 that overlap with convergent or divergent MACE tags from ref. 20. (c) Fly_modENCODE_MACE_tags. Fly PcG peaks from ref. 21 that overlap with convergent or divergent MACE tags from ref. 20 showing all MACE tags overlapping with each peak. (d) Fly_modENCODE_CAGE. Fly PcG peaks from ref. 21 that overlap with convergent or divergent CAGE tags from ref. 31. (e) Fly_modENCODE_CAGE_tags. Fly PcG peaks from ref. 21 that overlap with convergent or divergent CAGE tags from ref. 31 showing all CAGE tags overlapping with each peak. The source of tag data is listed as 'peaks' (indicating data from CAGE peaks in Supplemental Data File 4 of ref. 31), or 'file 3' (indicating RACE and other annotated transcript start sites from Supplemental Data File 3 of ref. 31). (f) Fly_Enderle_MACE. Fly PcG peaks from ref. 20 that overlap with convergent or divergent MACE tags from the same study. (g) Fly_Enderle_MACE_tags. Fly PcG peaks from ref. 20 that overlap with convergent or divergent MACE tags from the same study showing all MACE tags overlapping with each peak. (h) Fly_Enderle_CAGE. Fly PcG peaks from ref. 20 that overlap with convergent or divergent CAGE tags from ref. 31. (i) Fly_Enderle_CAGE_tags. Fly PcG peaks from ref. 20 that overlap with convergent or divergent CAGE tags from ref. 31 showing all CAGE tags overlapping with each peak.

Videos

  1. 1.

    Three-dimensional animation of vg mRNA showing downregulation upon noncoding RNA expression.

    The animation shows the three-dimensional reconstruction of the z stack from the double in situ hybridizations shown in Figure 3l-n. The animation shows a transgenic 3rd instar larval wing disc overexpressing the forward strand from pKC27vg.fwd′ under the control of the enGAL4 driver, which expresses in the posterior half of the disc. At the start of the animation, posterior is to the left, anterior is to the right. The domain of enGAL4 expression was defined in three dimensions using the noncoding RNA signal and is shown in green at the start of the animation, with the remaining ones shown in blue. As the animation plays, first the blue mask and then the green mask is removed, showing the in situ signals: green, forward noncoding RNA; red, vg mRNA; blue, DAPI. Subsequently, the noncoding RNA and DAPI channels are removed, leaving the vg mRNA signal in the red channel. The image is then rotated to show the downregulation of vg mRNA in the posterior part of the disc. See also Figure 3l-p.

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https://doi.org/10.1038/ng.3058

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