Article | Published:

A heterochromatin-dependent transcription machinery drives piRNA expression

Nature volume 549, pages 5459 (07 September 2017) | Download Citation

Abstract

Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters—small RNA source loci in animal gonads—is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    Transposable elements, epigenetics, and genome evolution. Science 338, 758–767 (2012)

  2. 2.

    & RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112 (2013)

  3. 3.

    & RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015)

  4. 4.

    , , & PIWI-interacting small RNAs: the vanguard of genome defence. Nat. Rev. Mol. Cell Biol. 12, 246–258 (2011)

  5. 5.

    & One loop to rule them all: the ping-pong cycle and piRNA-guided silencing. Trends Biochem. Sci. 41, 324–337 (2016)

  6. 6.

    , , & The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157, 1364–1379 (2014)

  7. 7.

    et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007)

  8. 8.

    et al. Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev. 28, 1667–1680 (2014)

  9. 9.

    et al. The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters. Cell 138, 1137–1149 (2009)

  10. 10.

    et al. The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing. Cell 157, 1353–1363 (2014)

  11. 11.

    , & Structural basis of transcription initiation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 129–143 (2015)

  12. 12.

    , , & Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561 (1989)

  13. 13.

    et al. TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation. Nature 465, 956–960 (2010)

  14. 14.

    et al. Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors. Mol. Cell 63, 97–109 (2016)

  15. 15.

    et al. CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell 151, 1488–1500 (2012)

  16. 16.

    & Direct recognition of initiator elements by a component of the transcription factor IID complex. Genes Dev. 8, 821–829 (1994)

  17. 17.

    , & TFIID sequence recognition of the initiator and sequences farther downstream in Drosophila class II genes. Genes Dev. 8, 830–842 (1994)

  18. 18.

    et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007)

  19. 19.

    , , & A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway. Mol. Cell 50, 749–761 (2013)

  20. 20.

    , , & Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830–836 (1996)

  21. 21.

    , , & Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 381, 127–134 (1996)

  22. 22.

    , , , & TBP-like factor is required for embryonic RNA polymerase II transcription in C. elegans. Mol. Cell 6, 715–722 (2000)

  23. 23.

    , , & The TBP-like factor CeTLF is required to activate RNA polymerase II transcription during C. elegans embryogenesis. Mol. Cell 6, 705–713 (2000)

  24. 24.

    et al. Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes. Mol. Cell. Biol. 26, 7492–7505 (2006)

  25. 25.

    , , , & TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Curr. Biol. 11, 282–287 (2001)

  26. 26.

    , & Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus. Science 290, 2312–2315 (2000)

  27. 27.

    et al. Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene. Mol. Cell 7, 509–515 (2001)

  28. 28.

    , , , & Spermiogenesis deficiency in mice lacking the Trf2 gene. Science 292, 1153–1155 (2001)

  29. 29.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  30. 30.

    et al. Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation. Genes Dev. 8, 2313–2323 (1994)

  31. 31.

    , , , & Transcription of histone gene cluster by differential core-promoter factors. Genes Dev. 21, 2936–2949 (2007)

  32. 32.

    et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006)

  33. 33.

    , , & RNA interference guides histone modification during the S phase of chromosomal replication. Curr. Biol. 18, 490–495 (2008)

  34. 34.

    et al. Cell cycle control of centromeric repeat transcription and heterochromatin assembly. Nature 451, 734–737 (2008)

  35. 35.

    et al. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498, 385–389 (2013)

  36. 36.

    , , & SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS Genet. 7, e1002195 (2011)

  37. 37.

    et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163, 445–455 (2015)

  38. 38.

    , , , & TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420, 439–445 (2002)

  39. 39.

    , , , & Transcriptional properties and splicing of the flamenco piRNA cluster. EMBO Rep. 15, 411–418 (2014)

  40. 40.

    et al. Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila. Genes Dev. 30, 840–855 (2016)

  41. 41.

    et al. UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery. Cell 151, 871–884 (2012)

  42. 42.

    , , & Drosophila TFIIA-S is up-regulated and required during Ras-mediated photoreceptor determination. Genes Dev. 10, 50–59 (1996)

  43. 43.

    et al. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat. Methods 8, 405–407 (2011)

  44. 44.

    , , , & Interaction of the COOH-terminal transactivation domain of p65 NF-κ B with TATA-binding protein, transcription factor IIB, and coactivators. J. Biol. Chem. 270, 7219–7226 (1995)

  45. 45.

    & A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA--promoter DNA complex formation. Genes Dev. 8, 995–1006 (1994)

  46. 46.

    , & A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly. Mol. Cell. Biol. 15, 6465–6473 (1995)

  47. 47.

    & Genes encoding Drosophila melanogaster RNA polymerase II general transcription factors: diversity in TFIIA and TFIID components contributes to gene-specific transcriptional regulation. J. Cell Biol. 150, F45–F50 (2000)

  48. 48.

    , , & TRF2 and the evolution of the bilateria. Genes Dev. 28, 2071–2076 (2014)

  49. 49.

    et al. Distinct functions of TBP and TLF/TRF2 during spermatogenesis: requirement of TLF for heterochromatic chromocenter formation in haploid round spermatids. Development 129, 945–955 (2002)

  50. 50.

    et al. Cleavage of TFIIA by Taspase1 activates TRF2-specified mammalian male germ cell programs. Dev. Cell 27, 188–200 (2013)

  51. 51.

    , , , & TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression. Sci. Rep. 6, 32069 (2016)

  52. 52.

    , & TIPT, a male germ cell-specific partner of TRF2, is chromatin-associated and interacts with HP1. Cell Cycle 7, 1415–1422 (2008)

  53. 53.

    et al. Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat. Methods 6, 431–434 (2009)

  54. 54.

    et al. The genetic makeup of the Drosophila piRNA pathway. Mol. Cell 50, 762–777 (2013)

  55. 55.

    , , & Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166, 1313–1321 (2004)

  56. 56.

    , & Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila. G3 (Bethesda) 4, 2279–2282 (2014)

  57. 57.

    , & Methods for homologous recombination in Drosophila. Methods Mol. Biol. 420, 155–174 (2008)

  58. 58.

    et al. MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra. J. Proteome Res. 13, 3679–3684 (2014)

  59. 59.

    et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015)

  60. 60.

    et al. Transcriptional regulators form diverse groups with context-dependent regulatory functions. Nature 528, 147–151 (2015)

  61. 61.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)

  62. 62.

    , & Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protocols 1, 729–748 (2006)

  63. 63.

    et al. Diversity and dynamics of the Drosophila transcriptome. Nature 512, 393–399 (2014)

  64. 64.

    . et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  65. 65.

    , , , & Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017)

  66. 66.

    et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010)

  67. 67.

    , , & Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 39, e141 (2011)

  68. 68.

    Accelerated profile HMM Searches. PLoS Comput. Biol. 7, e1002195 (2011)

  69. 69.

    et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

  70. 70.

    & Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinform. 9, 286–298 (2008)

  71. 71.

    , , , & Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009)

  72. 72.

    , & The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201 (2008)

  73. 73.

    & Analysis of compositionally biased regions in sequence databases. Methods Enzymol. 266, 554–571 (1996)

  74. 74.

    R Core Team. R: A language and environment for statistical computing (R Foundation for Statistical Computing, 2016)

  75. 75.

    ggplot2 (Springer, 2016)

  76. 76.

    Reshaping data with the reshape package. J. Stat. Softw. 21 (12), 1–20 (2007)

  77. 77.

    scales: scale functions for visualization. R package version 0.4.0. (2016)

  78. 78.

    M. preprocessCore: a collection of pre-processing functions. R package version 1.28.0. (2013)

  79. 79.

    et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002)

  80. 80.

    et al. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30, 1003–1005 (2014)

  81. 81.

    et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44 (D1), D447–D456 (2016)

Download references

Acknowledgements

We thank K. Meixner for experimental support, D. Handler and D. Jurczak for bioinformatics help, P. Duchek and J. Gokcezade for generating CRISPR-edited and transgenic flies, K. Mechtler and his team for mass spectrometry, T. Lendl for RNA FISH quantification, A. Schleiffer and M. Novatchkova for Moonshiner phylogenetic analysis, the Vienna Biocenter Core Facilities Next Generation Sequencing unit for deep sequencing, M. Elmaghraby for the Deadlock antigen, the Max F. Perutz Laboratories monoclonal facility for the Deadlock antibody, and the Vienna Drosophila RNAi Center, TRiP, and Bloomington stock centers for flies. We thank A. Ordonez, D. Handler, F. Mohn, F. Muerdter, M. Bühler, and especially O. Wueseke (Impulse Science) and Life Science Editors for comments on the manuscript. This work was supported by the Austrian Academy of Sciences and the European Community (ERC grant 260711EU and ERC-2015-CoG-682181). P.R.A. is supported by fellowships from the Alfred Benzon Foundation and the Novo Nordisk Foundation.

Author information

Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohrgasse 3, 1030 Vienna, Austria

    • Peter Refsing Andersen
    • , Laszlo Tirian
    • , Milica Vunjak
    •  & Julius Brennecke

Authors

  1. Search for Peter Refsing Andersen in:

  2. Search for Laszlo Tirian in:

  3. Search for Milica Vunjak in:

  4. Search for Julius Brennecke in:

Contributions

P.R.A. performed the experiments except the genetic bypass and promoter deletion experiments (both L.T.), and the S2 cell-based protein interaction assays (M.V.). P.R.A. and J.B. analysed the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Peter Refsing Andersen or Julius Brennecke.

Reviewer Information Nature thanks E. Brasset, T. Juven-Gershon and P. Zamore for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Notes 1-3 and Supplementary Figure 1, the uncropped western blot images.

  2. 2.

    Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    This file contains Drosophila melanogaster fly strains.

  2. 2.

    Supplementary Table 2

    This table contains oligo sequences.

  3. 3.

    Supplementary Table 3

    This table contains a list of antibodies used in the study.

  4. 4.

    Supplementary Table 4

    This table contains a list of plasmids used in the study.

  5. 5.

    Supplementary Table 5

    This table contains Stellaris probe sequences.

  6. 6.

    Supplementary Table 6

    This table contains sequence accessions for Extended Data Figure 2b

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature23482

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.