Abstract
RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21–30 nucleotide) RNAs1,2,3,4,5,6. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation7,8,9,10. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
Data deposits
Sequence data have been deposited in the DNA Data Bank of Japan under accession code DRA000418.
References
Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666 (2004)
Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004)
Kawamura, Y. et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793–797 (2008)
Ghildiyal, M. et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320, 1077–1081 (2008)
Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798–802 (2008)
Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803–806 (2008)
Allshire, R. C. & Karpen, G. H. Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nature Rev. Genet. 9, 923–937 (2008)
Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656–668 (2009)
Moazed, D. Small RNAs in transcriptional gene silencing and genome defence. Nature 457, 413–420 (2009)
Teixeira, F. K. et al. A role for RNAi in the selective correction of DNA methylation defects. Science 323, 1600–1604 (2009)
Llano, M. et al. Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. J. Mol. Biol. 360, 760–773 (2006)
Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M. C. Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19, 2837–2848 (2005)
Miyoshi, K., Okada, T. N., Siomi, H. & Siomi, M. C. Characterization of the miRNA–RISC loading complex and miRNA–RISC formed in the Drosophila miRNA pathway. RNA 15, 1282–1291 (2009)
Weeks, J. R., Hardin, S. E., Shen, J., Lee, J. M. & Greenleaf, A. L. Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev. 7, 2329–2344 (1993)
Lis, J. T. Imaging Drosophila gene activation and polymerase pausing in vivo. Nature 450, 198–202 (2007)
Wu, C. H. et al. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–1414 (2003)
Simon, J. A., Sutton, C. A., Lobell, R. B., Glaser, R. L. & Lis, J. T. Determinants of heat shock-induced chromosome puffing. Cell 40, 805–817 (1985)
Boehm, A. K., Saunders, A., Werner, J. & Lis, J. T. Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol. Cell. Biol. 23, 7628–7637 (2003)
Lee, C. et al. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. Mol. Cell. Biol. 28, 3290–3300 (2008)
Gilchrist, D. A. et al. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev. 22, 1921–1933 (2008)
Cai, W. et al. RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila. Development 135, 2917–2925 (2008)
Lim, D. H., Kim, J., Kim, S., Carthew, R. W. & Lee, Y. S. Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila. Biochem. Biophys. Res. Commun. 371, 525–530 (2008)
Kim, K., Lee, Y. S. & Carthew, R. W. Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes. RNA 13, 22–29 (2007)
Chambeyron, S. & Bickmore, W. A. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription. Genes Dev. 18, 1119–1130 (2004)
Kim, D. H., Villeneuve, L. M., Morris, K. V. & Rossi, J. J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nature Struct. Mol. Biol. 13, 793–797 (2006)
Kavi, H. H. & Birchler, J. A. Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila. Epigenetics Chromatin 2, 15 (2009)
El-Shami, M. et al. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev. 21, 2539–2544 (2007)
Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Rev. Genet. 10, 94–108 (2009)
Hoskins, R. A. et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res. 21, 182–192 (2011)
Lupo, R., Breiling, A., Bianchi, M. E. & Orlando, V. Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalise with Polycomb and maintain Fab-7 PRE silencing. Mol. Cell 7, 127–136 (2001)
Breiling, A., Turner, B. M., Bianchi, M. E. & Orlando, V. General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412, 651–655 (2001)
Stock, J. K. et al. Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nature Cell Biol. 9, 1428–1435 (2007)
Messmer, S., Franke, A. & Paro, R. Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster. Genes Dev. 6, 1241–1254 (1992)
Hsu, J. Y. et al. TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription. Genes Dev. 22, 2353–2358 (2008)
Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003)
Breiling, A., O’Neill, L. P., D’Eliseo, D., Turner, B. M. & Orlando, V. Epigenome changes in active and inactive Polycomb-group-controlled regions. EMBO Rep. 5, 976–982 (2004)
Pimpinelli, S., Bonaccorsi, S., Fanti, L. & Gatti, M. in Drosophila: A Laboratory Manual (eds Sullivan, W., Ashburner, M. & Hawley, S. ) 1–24 (Cold Spring Harbor Laboratory Press, 2000)
Corona, D. F., Armstrong, J. A. & Tamkun, J. W. Genetic and cytological analysis of Drosophila chromatin-remodeling factors. Methods Enzymol. 377, 70–85 (2004)
Cartwright, I. L. et al. Analysis of Drosophila chromatin structure in vivo. Methods Enzymol. 304, 462–496 (1999)
Kawano, M. et al. Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing. Biotechniques 49, 751–755 (2010)
de Hoon, M. J. et al. Cross-mapping and the identification of editing sites in mature microRNAs in high-throughput sequencing libraries. Genome Res. 20, 257–264 (2010)
Tweedie, S. et al. FlyBase: enhancing Drosophila Gene Ontology annotations. Nucleic Acids Res. 37, D555–D559 (2009)
Mituyama, T. Y. K. et al. The Functional RNA Database 3.0: databases to support mining and annotation of functional RNAs. Nucleic Acids Res. 37, D89–D92 (2009)
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)
Acknowledgements
We are deeply grateful to P. Macino for discussions. We also thank R. Carthew, E. Lai, Q. Liu, R. Paro, Y. Sik Lee, and J. T. Kadonaga for reagents. This work was supported by grants from the following: the National Institutes of Health (GM47477) to D.S.G.; the Deutsche Forschungsgemeinschaft (SPP 1356) to A.B.; the Japan Society for the Promotion of Science (JSPS) through the ‘Funding Program for Next Generation World-Leading Researchers’ (NEXT Program) (a Grant-in-Aid for Scientific Research (A) No. 20241047) and the Council for Science and Technology Policy to P.C.; the NEXT Program (a Research Grant-in-Aid to the RIKEN OSC) to K.M., M.C.S. and H.S.; Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency to M.C.S.; Fondazione Telethon, the Giovanni Armenise Harvard Foundation, FIRB-MIUR, Associazione Italiana Ricerca Cancro (AIRC), the Human Frontier Science Program CDA and the EMBO Young investigator program to D.F.V.C.; Fondazione Telethon, AIRC and the EU FP6 Epigenome Network of Excellence to V.O. This work was also made possible with the contribution of the Italian Ministry of Foreign Affairs, ‘Direzione Generale per la Promozione e la Cooperazione Culturale’ to V.O. A.S. is supported by a JSPS fellowship (ID P09745). Sequencing was provided by the Genas service (RIKEN Omics Science Center).
Author information
Authors and Affiliations
Contributions
F.M.C. and V.O. conceived the study. F.M.C. and A.B. performed the ChIP experiments. F.M.C. and K.M.P. carried out the chromatin fractionation assays and the western blotting. F.M.C. carried out the quantitative RT–PCR on S2 cells. K.M.P. and F.L.S. performed the quantitative RT–PCR on mutant larvae prepared by M.C.O. F.M.C. performed the co-immunoprecipitations and contributed reagents for the chromosome and permanganate footprinting experiments. M.C.O. performed the polytene chromosome experiments. G.O.K. and D.S.G. performed the permanganate footprinting experiments. A.M.B. performed the bioinformatic analysis. A.S. and P.C. performed the deep sequencing. K.M., H.S. and M.C.S. performed the purification of the AGO2-associated small RNAs. F.M.C., M.C.O., D.S.G., D.F.V.C. and V.O. designed the experiments and interpreted the results. F.M.C., D.S.G., D.F.V.C. and V.O. wrote the manuscript with the contribution of M.C.O., A.B. and A.M.B., as well as input from the other co-authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
The file contains Supplementary Figures 1-14 with legends, Supplementary Tables 1-4 and 6 (see separate file for table 5), a Supplementary Discussion and Supplementary References. (PDF 9375 kb)
Supplementary Table 5
The table shows tags mapping to heat shock loci. (XLS 4184 kb)
Rights and permissions
About this article
Cite this article
Cernilogar, F., Onorati, M., Kothe, G. et al. Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila. Nature 480, 391–395 (2011). https://doi.org/10.1038/nature10492
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature10492
This article is cited by
-
Aedes aegypti Argonaute 2 controls arbovirus infection and host mortality
Nature Communications (2023)
-
AGO2 silences mobile transposons in the nucleus of quiescent cells
Nature Structural & Molecular Biology (2023)
-
Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap-, Zeste-, Psq-, and Adf1-associated proteins
Cellular and Molecular Life Sciences (2022)
-
Action mechanisms and research methods of tRNA-derived small RNAs
Signal Transduction and Targeted Therapy (2020)
-
Various modes of HP1a interactions with the euchromatic chromosome arms in Drosophila ovarian somatic cells
Chromosoma (2020)
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.