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Nascent transcript sequencing visualizes transcription at nucleotide resolution

Abstract

Recent studies of transcription have revealed a level of complexity not previously appreciated even a few years ago, both in the intricate use of post-initiation control and the mass production of rapidly degraded transcripts. Dissection of these pathways requires strategies for precisely following transcripts as they are being produced. Here we present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that although promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus, nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.

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Figure 1: NET-seq visualizes active transcription via capture of 3′ RNA termini.
Figure 2: Observation of divergent transcripts reveals strong directionality at most promoters.
Figure 3: Rco1 suppresses antisense transcription at divergent promoters.
Figure 4: Frequent RNAPII pausing throughout gene bodies.
Figure 5: Dst1 relieves RNAPII pausing after backtracking.
Figure 6: Nucleosomes are a major barrier to transcription.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Raw sequencing data and processed data are available for download at http://www.ncbi.nlm.nih.gov/geo/ via GEO accession number GSE25107.

References

  1. Moore, M. J. & Proudfoot, N. J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009)

    CAS  Article  Google Scholar 

  2. Preker, P. et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science 322, 1851–1854 (2008)

    ADS  CAS  Article  Google Scholar 

  3. Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033–1037 (2009)

    ADS  CAS  Article  Google Scholar 

  4. Neil, H. et al. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast. Nature 457, 1038–1042 (2009)

    ADS  CAS  Article  Google Scholar 

  5. Rougvie, A. E. & Lis, J. T. The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell 54, 795–804 (1988)

    CAS  Article  Google Scholar 

  6. Proshkin, S., Rahmouni, A. R., Mironov, A. & Nudler, E. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 328, 504–508 (2010)

    ADS  CAS  Article  Google Scholar 

  7. Kassavetis, G. A. & Chamberlin, M. J. Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro . J. Biol. Chem. 256, 2777–2786 (1981)

    CAS  PubMed  Google Scholar 

  8. Shaevitz, J. W., Abbondanzieri, E. A., Landick, R. & Block, S. M. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003)

    ADS  CAS  Article  Google Scholar 

  9. Herbert, K. M. et al. Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125, 1083–1094 (2006)

    CAS  Article  Google Scholar 

  10. Hodges, C., Bintu, L., Lubkowska, L., Kashlev, M. & Bustamante, C. Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II. Science 325, 626–628 (2009)

    ADS  CAS  Article  Google Scholar 

  11. Kireeva, M. L. & Kashlev, M. Mechanism of sequence-specific pausing of bacterial RNA polymerase. Proc. Natl Acad. Sci. USA 106, 8900–8905 (2009)

    ADS  CAS  Article  Google Scholar 

  12. Kireeva, M. L. et al. Nature of the nucleosomal barrier to RNA polymerase II. Mol. Cell 18, 97–108 (2005)

    CAS  Article  Google Scholar 

  13. Kim, T. H. et al. A high-resolution map of active promoters in the human genome. Nature 436, 876–880 (2005)

    ADS  CAS  Article  Google Scholar 

  14. Lefrançois, P. et al. Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10, 37 (2009)

    Article  Google Scholar 

  15. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008)

    ADS  CAS  Article  Google Scholar 

  16. Rodríguez-Gil, A. et al. The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors. Nucl. Acids Res. 38, 4651–4664 (2010)

    Article  Google Scholar 

  17. Cai, H. & Luse, D. S. Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes. J. Biol. Chem. 262, 298–304 (1987)

    CAS  PubMed  Google Scholar 

  18. Bentley, D. R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008)

    ADS  CAS  Article  Google Scholar 

  19. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009)

    ADS  CAS  Article  Google Scholar 

  20. Markham, R. & Smith, J. D. The structure of ribonucleic acids. I. Cyclic nucleotides produced by ribonuclease and by alkaline hydrolysis. Biochem. J. 52, 552–557 (1952)

    CAS  Article  Google Scholar 

  21. Seila, A. C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008)

    ADS  CAS  Article  Google Scholar 

  22. Seila, A. C., Core, L. J., Lis, J. T. & Sharp, P. A. Divergent transcription: a new feature of active promoters. Cell Cycle 8, 2557–2564 (2009)

    CAS  Article  Google Scholar 

  23. Weiner, A., Hughes, A., Yassour, M., Rando, O. J. & Friedman, N. High-resolution nucleosome mapping reveals transcription-dependent promoter packaging. Genome Res. 20, 90–100 (2010)

    CAS  Article  Google Scholar 

  24. Pokholok, D. K. et al. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517–527 (2005)

    CAS  Article  Google Scholar 

  25. Carrozza, M. J. et al. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 123, 581–592 (2005)

    CAS  Article  Google Scholar 

  26. Keogh, M. C. et al. Cotranscriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell 123, 593–605 (2005)

    CAS  Article  Google Scholar 

  27. Li, B. et al. Histone H3 lysine 36 dimethylation (H3K36me2) is sufficient to recruit the Rpd3s histone deacetylase complex and to repress spurious transcription. J. Biol. Chem. 284, 7970–7976 (2009)

    CAS  Article  Google Scholar 

  28. Pinskaya, M., Gourvennec, S. & Morillon, A. H3 lysine 4 di- and tri-methylation deposited by cryptic transcription attenuates promoter activation. EMBO J. 28, 1697–1707 (2009)

    CAS  Article  Google Scholar 

  29. Govind, C. K. et al. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes. Mol. Cell 39, 234–246 (2010)

    CAS  Article  Google Scholar 

  30. Krogan, N. J. et al. Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23, 4207–4218 (2003)

    CAS  Article  Google Scholar 

  31. Nudler, E., Mustaev, A., Lukhtanov, E. & Goldfarb, A. The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 89, 33–41 (1997)

    CAS  Article  Google Scholar 

  32. Izban, M. G. & Luse, D. S. Factor-stimulated RNA polymerase II transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates. J. Biol. Chem. 267, 13647–13655 (1992)

    CAS  PubMed  Google Scholar 

  33. Reines, D., Conaway, R. C. & Conaway, J. W. Mechanism and regulation of transcriptional elongation by RNA polymerase II. Curr. Opin. Cell Biol. 11, 342–346 (1999)

    CAS  Article  Google Scholar 

  34. Kulish, D. & Struhl, K. TFIIS enhances transcriptional elongation through an artificial arrest site in vivo . Mol. Cell. Biol. 21, 4162–4168 (2001)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  36. Sigurdsson, S., Dirac-Svejstrup, A. B. & Svejstrup, J. Q. Evidence that transcript cleavage is essential for RNA polymerase II transcription and cell viability. Mol. Cell 38, 202–210 (2010)

    CAS  Article  Google Scholar 

  37. Li, B., Carey, M. & Workman, J. The role of chromatin during transcription. Cell 128, 707–719 (2007)

    CAS  Article  Google Scholar 

  38. Petesch, S. J. & Lis, J. T. Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134, 74–84 (2008)

    CAS  Article  Google Scholar 

  39. Kaplan, N. et al. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458, 362–366 (2009)

    ADS  CAS  Article  Google Scholar 

  40. Hall, M. A. et al. High-resolution dynamic mapping of histone-DNA interactions in a nucleosome. Nature Struct. Mol. Biol. 16, 124–129 (2009)

    CAS  Article  Google Scholar 

  41. Arigo, J. T., Eyler, D. E., Carroll, K. L. & Corden, J. L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. Mol. Cell 23, 841–851 (2006)

    CAS  Article  Google Scholar 

  42. Vasiljeva, L., Kim, M., Mutschler, H., Buratowski, S. & Meinhart, A. The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain. Nature Struct. Mol. Biol. 15, 795–804 (2008)

    CAS  Article  Google Scholar 

  43. Unrau, P. J. & Bartel, D. P. RNA-catalysed nucleotide synthesis. Nature 395, 260–263 (1998)

    ADS  CAS  Article  Google Scholar 

  44. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    Article  Google Scholar 

  45. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank C. Guthrie, N. Krogan, S. Luo, G. Schroth, J. Steitz and K. Yamamoto for advice and discussions; D. Breslow, P. Fordyce, A. Frost, J. Huff, M. Kampmann and M. Pufall for critical comments on the manuscript; C. Chu and N. Ingolia for help with sequencing and analysis; and S. Rouskin for help developing the ligation protocol. This research was supported by the Damon Runyon Cancer Research Foundation (DRG-1997-08 to L.S.C.) and by the Howard Hughes Medical Institute (to J.S.W.)

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L.S.C. and J.S.W. designed the experiments; L.S.C. performed the experiments and analysed the data; and L.S.C. and J.S.W. interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Jonathan S. Weissman.

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The authors declare no competing financial interests.

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Churchman, L., Weissman, J. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011). https://doi.org/10.1038/nature09652

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