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Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila

Nature volume 480, pages 391–395 (2011)Cite this article

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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.

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Figure 1: RNA Pol II promoter-proximal pausing on Hsp70 is decreased in cells treated with Dcr2 RNAi.
Figure 2: Chromatin localization of RNA Pol II and AGO2 after heat shock.
Figure 3: DCR2 and the RNAi effector protein AGO2 associate with RNA Pol II and NELF-E.
Figure 4: Features of AGO2-associated small RNAs.

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Data deposits

Sequence data have been deposited in the DNA Data Bank of Japan under accession code DRA000418.

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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

Author notes

  1. Filippo M. Cernilogar

    Present address: Present address: Helmholtz Center Munich, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Munich-Neuherberg, Germany.,

  2. Filippo M. Cernilogar and Maria Cristina Onorati: These authors contributed equally to this work.

Authors and Affiliations

  1. Dulbecco Telethon Institute, Epigenetics and Genome Reprogramming, IRCCS Fondazione Santa Lucia, Via del Fosso di Fiorano 64, 00143 Rome, Italy

    Filippo M. Cernilogar, Krishna Mohan Parsi, Federica Lo Sardo & Valerio Orlando

  2. Dulbecco Telethon Institute, IGB ABT-CNR, Via Pietro Castellino 111, 80131 Naples, Italy

    Filippo M. Cernilogar & Krishna Mohan Parsi

  3. Dipartimento STEMBIO, Dulbecco Telethon Institute at Università degli Studi di Palermo, Sezione Biologia Cellulare, Viale delle Scienze, Edificio 16, 90128 Palermo, Italy

    Maria Cristina Onorati & Davide F. V. Corona

  4. Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Greg O. Kothe & David S. Gilmour

  5. RIKEN Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

    A. Maxwell Burroughs, Alka Saxena & Piero Carninci

  6. Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

    Achim Breiling

  7. Keio University School of Medicine, Tokyo 160-8582, Japan

    Keita Miyoshi, Haruhiko Siomi & Mikiko C. Siomi

  8. JST, CREST, Saitama 332-0012, Japan

    Mikiko C. Siomi

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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.

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Correspondence to Valerio Orlando.

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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)

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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

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