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Nascent transcript sequencing visualizes transcription at nucleotide resolution

Nature volume 469, pages 368–373 (2011)Cite this article

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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.

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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.

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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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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco and California Institute for Quantitative Biosciences, San Francisco, 94158, California, USA

    L. Stirling Churchman & Jonathan S. Weissman

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  1. L. Stirling Churchman
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Contributions

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.

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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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