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PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution

Author

Listed:
  • Anniina Vihervaara

    (Science for Life Laboratory
    Cornell University)

  • Philip Versluis

    (Cornell University)

  • Samu V. Himanen

    (Science for Life Laboratory)

  • John T. Lis

    (Cornell University)

Abstract

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes.

Suggested Citation

  • Anniina Vihervaara & Philip Versluis & Samu V. Himanen & John T. Lis, 2023. "PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution," Nature Communications, Nature, vol. 14(1), pages 1-19, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42715-3
    DOI: 10.1038/s41467-023-42715-3
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    References listed on IDEAS

    as
    1. L. Stirling Churchman & Jonathan S. Weissman, 2011. "Nascent transcript sequencing visualizes transcription at nucleotide resolution," Nature, Nature, vol. 469(7330), pages 368-373, January.
    2. Anniina Vihervaara & Dig Bijay Mahat & Michael J. Guertin & Tinyi Chu & Charles G. Danko & John T. Lis & Lea Sistonen, 2017. "Transcriptional response to stress is pre-wired by promoter and enhancer architecture," Nature Communications, Nature, vol. 8(1), pages 1-16, December.
    3. Gregory T. Booth & Pabitra K. Parua & Miriam Sansó & Robert P. Fisher & John T. Lis, 2018. "Cdk9 regulates a promoter-proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast," Nature Communications, Nature, vol. 9(1), pages 1-10, December.
    4. Nadine Czudnochowski & Christian A. Bösken & Matthias Geyer, 2012. "Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition," Nature Communications, Nature, vol. 3(1), pages 1-12, January.
    5. Seychelle M. Vos & Lucas Farnung & Henning Urlaub & Patrick Cramer, 2018. "Structure of paused transcription complex Pol II–DSIF–NELF," Nature, Nature, vol. 560(7720), pages 601-606, August.
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