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Nascent RNA analyses: tracking transcription and its regulation

Nature Reviews Genetics volume 20, pages 705–723 (2019)Cite this article

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Abstract

The programmes that direct an organism’s development and maintenance are encoded in its genome. Decoding of this information begins with regulated transcription of genomic DNA into RNA. Although transcription and its control can be tracked indirectly by measuring stable RNAs, it is only by directly measuring nascent RNAs that the immediate regulatory changes in response to developmental, environmental, disease and metabolic signals are revealed. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide resolution, all of which have contributed novel insights into the mechanisms of gene regulation and transcription-coupled RNA processing. Here we critically evaluate the array of strategies used for investigating nascent transcription and discuss the recent conceptual advances they have provided.

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Fig. 1: The transcription cycle.
Fig. 2: Comparison of nascent RNA enrichment and sequencing assays.
Fig. 3: Imaging nascent RNA.
Fig. 4: Promoter-proximal pausing interferes with transcription initiation.
Fig. 5: Observing cleavage and polyadenylation in nascent RNA data sets.

References

  1. Liu, Y., Beyer, A. & Aebersold, R. On the dependency of cellular protein levels on mRNA abundance. Cell 165, 535–550 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Long, H. K., Prescott, S. L. & Wysocka, J. Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167, 1170–1187 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zabidi, M. A. & Stark, A. Regulatory enhancer-core-promoter communication via transcription factors and cofactors. Trends Genet. 32, 801–814 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Core, L. J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014). This study identifies the unified architecture of regulatory elements at genes and enhancers, comprising two core initiation regions to coordinate divergent transcription. It also maps enhancers across the human genome using their characteristic pattern of unstable, divergent transcription.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Scruggs, B. S. et al. Bidirectional transcription arises from two distinct hubs of transcription factor binding and active chromatin. Mol. Cell 58, 1101–1112 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ntini, E. et al. Polyadenylation site–induced decay of upstream transcripts enforces promoter directionality. Nat. Struct. Mol. Biol. 20, 923–928 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Li, W., Notani, D. & Rosenfeld, M. G. Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nat. Rev. Genet. 17, 207–223 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Chen, F. X., Smith, E. R. & Shilatifard, A. Born to run: control of transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 19, 464–478 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Zaborowska, J., Egloff, S. & Murphy, S. The pol II CTD: new twists in the tail. Nat. Struct. Mol. Biol. 23, 771–777 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Boettiger, A. N. & Levine, M. Synchronous and stochastic patterns of gene activation in the Drosophila embryo. Science 325, 471–473 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Henriques, T. et al. Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals. Mol. Cell 52, 517–528 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Mahat, D. B., Salamanca, H. H., Duarte, F. M., Danko, C. G. & Lis, J. T. Mammalian heat shock response and mechanisms underlying its genome-wide transcriptional regulation. Mol. Cell 62, 63–78 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rasmussen, E. B. & Lis, J. T. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl Acad. Sci. USA 90, 7923–7927 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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).

    Article  CAS  PubMed  Google Scholar 

  16. Vihervaara, A. et al. Transcriptional response to stress is pre-wired by promoter and enhancer architecture. Nat. Commun. 8, 255 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Williams, L. H. et al. Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks. Mol. Cell 58, 311–322 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vihervaara, A., Duarte, F. M. & Lis, J. T. Molecular mechanisms driving transcriptional stress responses. Nat. Rev. Genet. 19, 385–397 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vos, S. M. et al. Structure of activated transcription complex Pol II-DSIF-PAF-SPT6. Nature 560, 607–612 (2018). This article provides the atomic resolution cryogenic electron microscopy structure of the elongation-competent Pol II complex. It describes the changes in the composition and conformation of the transcription complex as it proceeds from pause into elongation.

    Article  CAS  PubMed  Google Scholar 

  20. Vos, S. M., Farnung, L., Urlaub, H. & Cramer, P. Structure of paused transcription complex Pol II–DSIF–NELF. Nature 560, 601–606 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wu, C.-H. et al. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–1414 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fuda, N. J., Ardehali, M. B. & Lis, J. T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461, 186–192 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tian, B. & Graber, J. H. Signals for pre-mRNA cleavage and polyadenylation. Wiley Interdiscip. Rev. RNA 3, 385–396 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Proudfoot, N. J. Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut. Science 352, aad9926 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Yudkovsky, N., Ranish, J. A. & Hahn, S. A transcription reinitiation intermediate that is stabilized by activator. Nature 408, 225 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Mandal, S. S. et al. Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II. Proc. Natl Acad. Sci. USA 101, 7572–7577 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. McCracken, S. et al. 5’-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 3306–3318 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Shimotohno, K., Kodama, Y., Hashimoto, J. & Miura, K. I. Importance of 5’-terminal blocking structure to stabilize mRNA in eukaryotic protein synthesis. Proc. Natl Acad. Sci. USA 74, 2734–2738 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wen, Y. & Shatkin, A. J. Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev. 13, 1774–1779 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Naftelberg, S., Schor, I. E., Ast, G. & Kornblihtt, A. R. Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu. Rev. Biochem. 84, 165–198 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Miller, O. L. & Beatty, B. R. Visualization of nucleolar genes. Science 164, 955–957 (1969).

    Article  PubMed  Google Scholar 

  32. Miller, O. L., Hamkalo, B. A. & Thomas, C. A. Visualization of bacterial genes in action. Science 169, 392–395 (1970).

    Article  PubMed  Google Scholar 

  33. Pelling, C. Ribonukleinsäure-Synthese der Riesenchromosomen. Chromosoma 15, 71–122 (1964).

    Article  CAS  PubMed  Google Scholar 

  34. Bernecky, C., Herzog, F., Baumeister, W., Plitzko, J. M. & Cramer, P. Structure of transcribing mammalian RNA polymerase II. Nature 529, 551–554 (2016).

    Article  CAS  PubMed  Google Scholar 

  35. Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 ångstrom resolution. Science 292, 1863–1876 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Bhatt, D. M. et al. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions. Cell 150, 279–290 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Henikoff, S., Henikoff, J. G., Sakai, A., Loeb, G. B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460–469 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mayer, A. et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 161, 541–554 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Weber, C. M., Ramachandran, S. & Henikoff, S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol. Cell 53, 819–830 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011). This article introduces NET-seq, which was performed in S. cerevisiae by immunoprecipitating epitope-tagged Pol II.

    Article  CAS  PubMed  Google Scholar 

  41. Nojima, T. et al. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015). This study applies NET-seq to human cells using antibodies to Ser5-phosphorylated, Ser2-phosphorylated and unphosphorylated Pol II CTD, demonstrating the positions of different Pol II modifications genome-wide.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kwak, H., Fuda, N. J., Core, L. J. & Lis, J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013). This article describes PRO-seq, which maps active Pol II positions at single-nucleotide resolution genome-wide.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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). This study describes Start-seq, which was used to identify initiating and promoter-proximal pausing positions across the D. melanogaster genome.

    Article  CAS  PubMed  Google Scholar 

  44. Fuchs, G. et al. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol. 15, R69 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Herzog, V. A. et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat. Methods 14, 1198–1204 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Schofield, J. A., Duffy, E. E., Kiefer, L., Sullivan, M. C. & Simon, M. D. TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding. Nat. Methods 15, 221–225 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Schwalb, B. et al. TT-seq maps the human transient transcriptome. Science 352, 1225–1228 (2016). This article describes TT-seq, which uses short metabolic labeling times to capture newly transcribed RNAs in human cells.

    Article  CAS  PubMed  Google Scholar 

  48. 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). This article describes GRO-seq and demonstrates widespread promoter-proximal pausing and bidirectional transcription across the human genome.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tome, J. M., Tippens, N. D. & Lis, J. T. Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers. Nat. Genet. 50, 1533–1541 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Booth, G. T., Parua, P. K., Sansó, M., Fisher, R. P. & Lis, J. T. Cdk9 regulates a promoter-proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast. Nat. Commun. 9, 543 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Duarte, F. M. et al. Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation. Genes Dev. 30, 1731–1746 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hah, N. et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kaikkonen, M. U. et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell 51, 310–325 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Levesque, M. J. & Raj, A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat. Methods 10, 246–248 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Shah, S. et al. Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH. Cell 174, 363–376 (2018). The imaging technique described in this article allows visualization of the synthesis of more than 10,000 genes in single cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lim, B. Imaging transcriptional dynamics. Curr. Opin. Biotechnol. 52, 49–55 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wuarin, J. & Schibler, U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol. Cell. Biol. 14, 7219–7225 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Hsiao, Y.-H. E. et al. RNA editing in nascent RNA affects pre-mRNA splicing. Genome Res. 28, 812–823 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Ke, S. et al. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 31, 990–1006 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Pandya-Jones, A. et al. Splicing kinetics and transcript release from the chromatin compartment limit the rate of lipid A-induced gene expression. RNA 19, 811–827 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Johnson, D. S., Mortazavi, A., Myers, R. M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. Townley-Tilson, W. H. D., Pendergrass, S. A., Marzluff, W. F. & Whitfield, M. L. Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein. RNA 12, 1853–1867 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Nojima, T. et al. RNA polymerase II phosphorylated on CTD serine 5 interacts with the spliceosome during co-transcriptional splicing. Mol. Cell 72, 369–379 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Schlackow, M. et al. Distinctive patterns of transcription and RNA processing for human lincRNAs. Mol. Cell 65, 25–38 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Henriques, T. et al. Widespread transcriptional pausing and elongation control at enhancers. Genes Dev. 32, 26–41 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Gariglio, P., Buss, J. & Green, M. H. Sarkosyl activation of RNA polymerase activity in mitotic mouse cells. FEBS Lett 44, 330–333 (1974).

    Article  CAS  PubMed  Google Scholar 

  67. Smale, S. T. Nuclear run-on assay. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot5329 (2009).

    Article  PubMed  Google Scholar 

  68. Core, L. J. et al. Defining the status of RNA polymerase at promoters. Cell Rep 2, 1025–1035 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Adelman, K. & Lis, J. T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Min, I. M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Li, J. et al. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing. Mol. Cell 50, 711–722 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Pugh, B. F. & Venters, B. J. Genomic organization of human transcription initiation complexes. PLOS ONE 11, e0149339 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Gressel, S. et al. CDK9-dependent RNA polymerase II pausing controls transcription initiation. eLife 6, e29736 (2017). This article demonstrates the worth of combining multiple techniques to investigate transcription. The study uses TT-seq, mNET-seq, and structural modelling to examine the interplay of Pol II initiation and pausing.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Chu, T. et al. Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme. Nat. Genet. 50, 1553–1564 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Dölken, L. et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–1972 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Jürges, C., Dölken, L. & Erhard, F. Dissecting newly transcribed and old RNA using GRAND-SLAM. Bioinformatics 34, i218–i226 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Kenzelmann, M. et al. Microarray analysis of newly synthesized RNA in cells and animals. Proc. Natl Acad. Sci. USA 104, 6164–6169 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Muhar, M. et al. SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science 360, 800–805 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Rabani, M. et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat. Biotechnol. 29, 436–442 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Rabani, M. et al. High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies. Cell 159, 1698–1710 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Paulsen, M. T. et al. Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc. Natl Acad. Sci. USA 110, 2240–2245 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Akbalik, G. et al. Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry. RNA Biol. 14, 20–28 (2017).

    Article  PubMed  Google Scholar 

  83. Jao, C. Y., & Salic, A. Exploring, R. N. A. transcription and turnover in vivo by using click chemistry. Proc. Natl Acad. Sci. USA 105, 15779–15784 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Nyhan, W. L. Nucleotide synthesis via salvage pathway. eLS https://doi.org/10.1038/npg.els.0001399 (2001).

  85. Stoeckius, M., Grün, D. & Rajewsky, N. Paternal RNA contributions in the Caenorhabditis elegans zygote. EMBO J. 33, 1740–1750 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Sidaway-Lee, K., Costa, M. J., Rand, D. A., Finkenstadt, B. & Penfield, S. Direct measurement of transcription rates reveals multiple mechanisms for configuration of the Arabidopsis ambient temperature response. Genome Biol. 15, R45 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  87. Bauman, J. G., Wiegant, J., Borst, P. & van Duijn, P. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA. Exp. Cell Res. 128, 485–490 (1980).

    Article  CAS  PubMed  Google Scholar 

  88. Gall, J. G. & Pardue, M. L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl Acad. Sci. USA 63, 378–383 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Singer, R. H. & Ward, D. C. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl Acad. Sci. USA 79, 7331–7335 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Vargas, D. Y., Raj, A., Marras, S. A. E., Kramer, F. R. & Tyagi, S. Mechanism of mRNA transport in the nucleus. Proc. Natl Acad. Sci. USA 102, 17008–17013 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    Article  CAS  PubMed  Google Scholar 

  92. Yunger, S., Rosenfeld, L., Garini, Y. & Shav-Tal, Y. Single-allele analysis of transcription kinetics in living mammalian cells. Nat. Methods 7, 631–633 (2010).

    Article  CAS  PubMed  Google Scholar 

  93. Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A. & Singer, R. H. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Lionnet, T. et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat. Methods 8, 165–170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Germier, T. et al. Real-time imaging of a single gene reveals transcription-initiated local confinement. Biophys. J. 113, 1383–1394 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Tantale, K. et al. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat. Commun. 7, 12248 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Larsson, A. J. M. et al. Genomic encoding of transcriptional burst kinetics. Nature 565, 251–254 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Gebhardt, J. C. M. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat. Methods 10, 421–426 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Liu, Z. & Tjian, R. Visualizing transcription factor dynamics in living cells. J. Cell Biol. 217, 1181–1191 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Gu, B. et al. Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements. Science 359, 1050–1055 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Bartman, C. R., Hsu, S. C., Hsiung, C. C.-S., Raj, A. & Blobel, G. A. Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Mol. Cell 62, 237–247 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Fukaya, T., Lim, B. & Levine, M. Enhancer control of transcriptional bursting. Cell 166, 358–368 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Mir, M. et al. Dense Bicoid hubs accentuate binding along the morphogen gradient. Genes Dev. 31, 1784–1794 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Adelman, K. et al. Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17, 103–112 (2005).

    Article  CAS  PubMed  Google Scholar 

  107. Azofeifa, J. G. et al. Enhancer RNA profiling predicts transcription factor activity. Genome Res. 28, 334–344 (2018). This study uses computational tools to identify enhancers from RO-seq data and demonstrates that these data can identify active transcription factor motifs.

    Article  CAS  PubMed Central  Google Scholar 

  108. Danko, C. G. et al. Identification of active transcriptional regulatory elements from GRO-seq data. Nat. Methods 12, 433–438 (2015). This study identifies enhancers and promoters from run-on data by identifying sites of bidirectional transcription.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Shivram, H. & Iyer, V. R. Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies. RNA 24, 1266–1274 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Gilmour, D. S. & Lis, J. T. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. Mol. Cell. Biol. 6, 3984–3989 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Krumm, A., Meulia, T., Brunvand, M. & Groudine, M. The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. Genes Dev. 6, 2201–2213 (1992).

    Article  CAS  PubMed  Google Scholar 

  112. Meininghaus, M. & Eick, D. Requirement of the carboxy-terminal domain of RNA polymerase II for the transcriptional activation of chromosomal c-fos and hsp70A genes. FEBS Lett 446, 173–176 (1999).

    Article  CAS  PubMed  Google Scholar 

  113. Plet, A., Eick, D. & Blanchard, J. M. Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns. Oncogene 10, 319–328 (1995).

    CAS  PubMed  Google Scholar 

  114. Danko, C. G. et al. Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells. Mol. Cell 50, 212–222 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Muse, G. W. et al. RNA polymerase is poised for activation across the genome. Nat. Genet. 39, 1507–1511 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Zeitlinger, J. et al. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat. Genet. 39, 1512–1516 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Adelman, K. et al. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc. Natl Acad. Sci. USA 106, 18207–18212 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Donner, A. J., Ebmeier, C. C., Taatjes, D. J. & Espinosa, J. M. CDK8 is a positive regulator of transcriptional elongation within the serum response network. Nat. Struct. Mol. Biol. 17, 194–201 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Dukler, N. et al. Nascent RNA sequencing reveals a dynamic global transcriptional response at genes and enhancers to the natural medicinal compound celastrol. Genome Res. 27, 1816–1829 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Escoubet-Lozach, L. et al. Mechanisms establishing TLR4-responsive activation states of inflammatory response genes. PLOS Genet. 7, e1002401 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Galbraith, M. D. et al. HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia. Cell 153, 1327–1339 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Titov, D. V. et al. XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nat. Chem. Biol. 7, 182–188 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Vispé, S. et al. Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA. Mol. Cancer Ther. 8, 2780–2790 (2009).

    Article  PubMed  CAS  Google Scholar 

  126. Chao, S.-H. & Price, D. H. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J. Biol. Chem. 276, 31793–31799 (2001).

    Article  CAS  PubMed  Google Scholar 

  127. Jonkers, I., Kwak, H. & Lis, J. T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. eLife 3, e02407 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  128. Traut, T. W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994).

    Article  CAS  PubMed  Google Scholar 

  129. Ashe, H. L., Monks, J., Wijgerde, M., Fraser, P. & Proudfoot, N. J. Intergenic transcription and transinduction of the human beta-globin locus. Genes Dev. 11, 2494–2509 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Routledge, S. J. E. & Proudfoot, N. J. Definition of transcriptional promoters in the human beta globin locus control region. J. Mol. Biol. 323, 601–611 (2002).

    Article  CAS  PubMed  Google Scholar 

  131. Kim, H. S. et al. Pluripotency factors functionally premark cell-type-restricted enhancers in ES cells. Nature 556, 510–514 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Tippens, N. D., Vihervaara, A. & Lis, J. T. Enhancer transcription: what, where, when, and why? Genes Dev. 32, 1–3 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Schaukowitch, K. et al. Enhancer RNA facilitates NELF release from immediate early genes. Mol. Cell 56, 29–42 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Chen, F. X. et al. PAF1 regulation of promoter-proximal pause release via enhancer activation. Science 357, 1294–1298 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Galli, G. G. et al. YAP drives growth by controlling transcriptional pause release from dynamic enhancers. Mol. Cell 60, 328–337 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. McKnight, S. L. & Miller, O. L. Post-replicative nonribosomal transcription units in D. melanogaster embryos. Cell 17, 551–563 (1979).

    Article  CAS  PubMed  Google Scholar 

  137. Chubb, J. R., Trcek, T., Shenoy, S. M. & Singer, R. H. Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Golding, I., Paulsson, J., Zawilski, S. M. & Cox, E. C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  PubMed  Google Scholar 

  139. Lenstra, T. L., Coulon, A., Chow, C. C. & Larson, D. R. Single-molecule imaging reveals a switch between spurious and functional ncRNA transcription. Mol. Cell 60, 597–610 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Bothma, J. P. et al. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc. Natl Acad. Sci. USA 111, 10598–10603 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Chen, L.-F. et al. Enhancer histone acetylation modulates transcriptional bursting dynamics of neuronal activity-inducible genes. Cell Rep. 26, 1174–1188 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Bartman, C. R. et al. Transcriptional burst initiation and polymerase pause release are key control points of transcriptional regulation. Mol. Cell 73, 519–532 (2019).

    Article  CAS  PubMed  Google Scholar 

  143. Chen, F., Gao, X. & Shilatifard, A. Stably paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor triptolide. Genes Dev. 29, 39–47 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  144. Core, L. & Adelman, K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes Dev. https://doi.org/10.1101/gad.325142.119 (2019).

    Article  CAS  Google Scholar 

  145. Buckley, M. S., Kwak, H., Zipfel, W. R. & Lis, J. T. Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation. Genes Dev. 28, 14–19 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Steurer, B. et al. Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA polymerase II. Proc. Natl Acad. Sci. USA 115, E4368–E4376 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Krebs, A. R. et al. Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters. Mol. Cell 67, 411–422.e4 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Moore, M. J. From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).

    Article  CAS  PubMed  Google Scholar 

  149. Singh, G., Pratt, G., Yeo, G. W. & Moore, M. J. The clothes make the mRNA: past and present trends in mRNP fashion. Annu. Rev. Biochem. 84, 325–354 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Bass, B. L. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846 (2002).

    Article  CAS  PubMed  Google Scholar 

  151. Meyer, K. D. & Jaffrey, S. R. Rethinking m6A readers, writers, and erasers. Annu. Rev. Cell Dev. Biol. 33, 319–342 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Merkhofer, E. C., Hu, P. & Johnson, T. L. Introduction to cotranscriptional RNA splicing. Methods Mol. Biol. Clifton NJ 1126, 83–96 (2014).

    Article  CAS  Google Scholar 

  153. Osheim, Y. N., O.L. Miller, Jr & Beyer, A. L. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell 43, 143–151 (1985).

    Article  CAS  PubMed  Google Scholar 

  154. Görnemann, J., Kotovic, K. M., Hujer, K. & Neugebauer, K. M. Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex. Mol. Cell 19, 53–63 (2005).

    Article  PubMed  CAS  Google Scholar 

  155. Lacadie, S. A. & Rosbash, M. Cotranscriptional spliceosome assembly dynamics and the role of U1 snRNA:5’ss base pairing in yeast. Mol. Cell 19, 65–75 (2005).

    Article  CAS  PubMed  Google Scholar 

  156. Listerman, I., Sapra, A. K. & Neugebauer, K. M. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat. Struct. Mol. Biol. 13, 815–822 (2006).

    Article  CAS  PubMed  Google Scholar 

  157. Keren, H., Lev-Maor, G. & Ast, G. Alternative splicing and evolution: diversification, exon definition and function. Nat. Rev. Genet. 11, 345–355 (2010).

    Article  CAS  PubMed  Google Scholar 

  158. Herzel, L., Ottoz, D. S. M., Alpert, T. & Neugebauer, K. M. Splicing and transcription touch base: co-transcriptional spliceosome assembly and function. Nat. Rev. Mol. Cell Biol. 18, 637–650 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. Carrillo Oesterreich, F. et al. Splicing of nascent RNA coincides with intron exit from RNA polymerase II. Cell 165, 372–381 (2016).

    Article  CAS  Google Scholar 

  160. Herzel, L., Straube, K. & Neugebauer, K. M. Long-read sequencing of nascent RNA reveals coupling among RNA processing events. Genome Res. 28, 1008–1019 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Prabhala, G., Rosenberg, G. H. & Käufer, N. F. Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe. Yeast 8, 171–182 (1992).

    Article  CAS  PubMed  Google Scholar 

  162. Pai, A. A. et al. The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture. eLife 6, e32537 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  163. Khodor, Y. L. et al. Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila. Genes Dev. 25, 2502–2512 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Louloupi, A., Ntini, E., Conrad, T. & Ørom, U. A. V. Transient N-6-methyladenosine transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency. Cell Rep 23, 3429–3437 (2018).

    Article  CAS  PubMed  Google Scholar 

  165. Eisenberg, E., & Levanon, E. Y. A-to-I, R. N. A. editing — immune protector and transcriptome diversifier. Nat. Rev. Genet. 19, 473–490 (2018).

    Article  CAS  PubMed  Google Scholar 

  166. Wang, I. X. et al. RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II. Cell Rep 6, 906–915 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Rodriguez, J., Menet, J. S. & Rosbash, M. Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila. Mol. Cell 47, 27–37 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Eaton, J. D. et al. Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity. Genes Dev. 32, 127–139 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. Chiu, A. C. et al. Transcriptional pause sites delineate stable nucleosome-associated premature polyadenylation suppressed by U1 snRNP. Mol. Cell 69, 648–663 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  170. Tian, B. & Manley, J. L. Alternative cleavage and polyadenylation: the long and short of it. Trends Biochem. Sci. 38, 312–320 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. Vilborg, A., Passarelli, M. C., Yario, T. A., Tycowski, K. T. & Steitz, J. A. Widespread inducible transcription downstream of human genes. Mol. Cell 59, 449–461 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Vilborg, A. et al. Comparative analysis reveals genomic features of stress-induced transcriptional readthrough. Proc. Natl Acad. Sci. USA 114, E8362–E8371 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Lai, F., Gardini, A., Zhang, A. & Shiekhattar, R. Integrator mediates the biogenesis of enhancer RNAs. Nature 525, 399–403 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. O’Reilly, D. et al. Human snRNA genes use polyadenylation factors to promote efficient transcription termination. Nucleic Acids Res 42, 264–275 (2014).

    Article  PubMed  CAS  Google Scholar 

  175. Ha, M. & Kim, V. N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 15, 509–524 (2014).

    Article  CAS  PubMed  Google Scholar 

  176. Dhir, A., Dhir, S., Proudfoot, N. J. & Jopling, C. L. Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs. Nat. Struct. Mol. Biol. 22, 319–327 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  177. Jonkers, I. & Lis, J. T. Getting up to speed with transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 167–177 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  178. Venkatesh, S. & Workman, J. L. Histone exchange, chromatin structure and the regulation of transcription. Nat. Rev. Mol. Cell Biol. 16, 178–189 (2015).

    Article  CAS  PubMed  Google Scholar 

  179. Descostes, N. et al. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells. eLife 3, e02105 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  180. Hintermair, C. et al. Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation. EMBO J 31, 2784–2797 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. Harlen, K. M. et al. Comprehensive RNA polymerase II interactomes reveal distinct and varied roles for each phospho-CTD residue. Cell Rep 15, 2147–2158 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. Gates, L. A., Foulds, C. E. & O’Malley, B. W. Histone marks in the ‘driver’s seat’: Functional roles in steering the transcription cycle. Trends Biochem. Sci. 42, 977–989 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. Klymenko, T. & Müller, J. The histone methyltransferases trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins. EMBO Rep. 5, 373–377 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  184. Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58–69 (2007).

    Article  CAS  PubMed  Google Scholar 

  185. Sims, R. J. et al. Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol. Cell 28, 665–676 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  186. Bieberstein, N. I., Carrillo Oesterreich, F., Straube, K. & Neugebauer, K. M. First exon length controls active chromatin signatures and transcription. Cell Rep 2, 62–68 (2012).

    Article  CAS  PubMed  Google Scholar 

  187. Huff, J. T., Plocik, A. M., Guthrie, C. & Yamamoto, K. R. Reciprocal intronic and exonic histone modification regions in humans. Nat. Struct. Mol. Biol. 17, 1495–1499 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  188. Andersson, R., Enroth, S., Rada-Iglesias, A., Wadelius, C. & Komorowski, J. Nucleosomes are well positioned in exons and carry characteristic histone modifications. Genome Res. 19, 1732–1741 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  189. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  191. Kolasinska-Zwierz, P. et al. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat. Genet. 41, 376–381 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Venkatesh, S. et al. Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes. Nature 489, 452–455 (2012).

    Article  CAS  PubMed  Google Scholar 

  193. McDaniel, S. L. & Strahl, B. D. Shaping the cellular landscape with Set2/SETD2 methylation. Cell. Mol. Life Sci. 74, 3317–3334 (2017).

    Article  CAS  PubMed  Google Scholar 

  194. Ernst, J. & Kellis, M. Discovery and characterization of chromatin states for systematic annotation of the human genome. Nat. Biotechnol. 28, 817–825 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  195. Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  196. Arnold, C. D. et al. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339, 1074–1077 (2013).

    Article  CAS  PubMed  Google Scholar 

  197. Mikhaylichenko, O. et al. The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev. 32, 42–57 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  198. Andersson, R., Sandelin, A. & Danko, C. G. A unified architecture of transcriptional regulatory elements. Trends Genet. 31, 426–433 (2015).

    Article  CAS  PubMed  Google Scholar 

  199. Dao, L. T. M. et al. Genome-wide characterization of mammalian promoters with distal enhancer functions. Nat. Genet. 49, 1073–1081 (2017).

    Article  CAS  PubMed  Google Scholar 

  200. Natsume, T. & Kanemaki, M. T. Conditional degrons for controlling protein expression at the protein level. Annu. Rev. Genet. 51, 83–102 (2017).

    Article  CAS  PubMed  Google Scholar 

  201. Ozer, A., Pagano, J. M. & Lis, J. T. New technologies provide quantum changes in the scale, speed, and success of SELEX methods and aptamer characterization. Mol. Ther. Nucleic Acids 3, e183 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  202. Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72 (2019).

    Article  CAS  PubMed  Google Scholar 

  203. Almada, A. E., Wu, X., Kriz, A. J., Burge, C. B. & Sharp, P. A. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499, 360–363 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  204. Mayran, A. & Drouin, J. Pioneer transcription factors shape the epigenetic landscape. J. Biol. Chem. 293, 13795–13804 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  205. Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  206. Kadonaga, J. T. Perspectives on the RNA polymerase II core promoter. Wiley Interdiscip. Rev. Dev. Biol. 1, 40–51 (2012).

    Article  CAS  PubMed  Google Scholar 

  207. Schilbach, S. et al. Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature 551, 204–209 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  208. Conaway, R. C. & Conaway, J. W. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters. Proc. Natl Acad. Sci. USA 86, 7356–7360 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  209. Tirode, F., Busso, D., Coin, F. & Egly, J. M. Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7. Mol. Cell 3, 87–95 (1999).

    Article  CAS  PubMed  Google Scholar 

  210. Hsin, J.-P. & Manley, J. L. The RNA polymerase II CTD coordinates transcription and RNA processing. Genes Dev. 26, 2119–2137 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  211. Fitz, J., Neumann, T. & Pavri, R. Regulation of RNA polymerase II processivity by Spt5 is restricted to a narrow window during elongation. EMBO J. 37, e97965 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  212. Takeuchi, A. et al. Loss of Sfpq causes long-gene transcriptopathy in the brain. Cell Rep 23, 1326–1341 (2018).

    Article  CAS  PubMed  Google Scholar 

  213. Mueller, B. et al. Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction. Genes Dev. 31, 451–462 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  214. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  215. Kristjánsdóttir, K. et al. Population-scale study of eRNA transcription reveals bipartite functional enhancer architecture. Preprint at bioRxiv https://doi.org/10.1101/426908 (2018).

  216. Michel, M. et al. TT-seq captures enhancer landscapes immediately after T-cell stimulation. Mol. Syst. Biol. 13, 920 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  217. Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  218. ENCODE Project Consortium. A user’s guide to the Encyclopedia of DNA Elements (ENCODE). PLOS Biol. 9, e1001046 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank members of the Lis laboratory for insightful discussions. They also thank the reviewers for their invaluable comments. This work was supported by the National Institute of General Medical Sciences grants R01-GM025232 (J.T.L.), F32-GM129904 (E.M.W.) and T32-HD057854 (N.D.T), and the Sigrid Jusélius Foundation (A.V.).

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  1. These authors contributed equally: Erin M. Wissink, Anniina Vihervaara.

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  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Erin M. Wissink, Anniina Vihervaara, Nathaniel D. Tippens & John T. Lis

  2. Tri-Institutional Training Program in Computational Biology and Medicine, New York, NY, USA

    Nathaniel D. Tippens

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Glossary

Enhancer RNAs

(eRNAs). Short (50–2000-nucleotide) non-coding RNAs that are produced by RNA polymerase II from enhancers. The production of enhancer RNAs moderately correlates with the functional activity of the enhancer.

Core initiation regions

Short (~60-nucleotide) regions at promoter and enhancer transcription start sites that provide a binding platform for general transcription factors. At promoters, ‘core promoter’ is used as a synonym for ‘core initiation region’.

C-terminal domain (CTD) of the RBP1 subunit of Pol II

This domain contains multiple repeats of seven amino acids and serves as a flexible binding scaffold for transcriptional regulators. The post-translational modifications of the heptad repeat greatly influence the regulatory interactions and, therefore, transcriptional processes throughout the cycle.

Nascent RNA

RNA that is in the process of being synthesized via transcription.

Divergent transcripts

In metazoans, genes and enhancers drive transcription from two core initiation regions from both strands in opposing directions. In genes, the mRNA-coding strand is termed the ‘sense strand’, and the antisense strand produces the divergent transcript.

Chromatin immunoprecipitation followed by sequencing (ChIP–seq)

A method in which protein–DNA interactions are stabilized, chromatin is sheared and fragments with a protein of interest are enriched using an antibody. Purified DNA from the enriched fragments is sequenced, providing genome-wide maps of protein localization.

RNA immunoprecipitation followed by sequencing

A technique that is used to identify which RNAs interact with a given protein. It uses antibody-mediated enrichment of a protein, after which its interacting RNAs can be isolated, reverse-transcribed and sequenced.

Paired-end sequencing

High-throughput sequencing of DNA molecules from both ends, which provides information from 3′ and 5′ ends of each DNA fragment, and allows more accurate mapping of the reads to the reference genome.

Permanganate treatment and chromatin immunoprecipitation followed by sequencing (PIP–seq)

A technique that uses permanganate, which oxidizes unpaired thymines in DNA, to detect the exact locations of open transcription complexes across the genome. RNA polymerase II is then immunoprecipitated, and DNA that has been oxidized by permanganate is cleaved using piperidine. These cleaved sites are identified by high-throughput sequencing.

Transcriptional bursting

Transcription occurs infrequently, and when a gene is turned on, many polymerases transcribe many copies of mRNA in a short time, which is known as transcriptional bursting. Bursting at a given gene is characterized by the duration, amplitude and frequency of transcription.

Cryogenic electron microscopy

An electron microscopy technique that visualizes molecules at cryogenic (−200 °C) temperatures. It allows near atomic resolution (less than 4 Å) imaging of complex molecules and molecule complexes in their native conformation without crystallization or embedding of the sample.

Intron definition

A splicing model in which specific sequences that demarcate introns are sufficient for spliceosomes to recognize intron boundaries.

Exon definition

A model by which proteins that bind to exons are required for the spliceosome to recognize sequences that demarcate introns.

R-loops

Three-stranded DNA–RNA hybrid structures formed, for example, by template DNA, the complement nascent RNA and the non-template single-stranded DNA.

Cap analysis of gene expression

A technique that measures RNA expression and maps transcription start sites of gene promoters. It provides precise maps of transcription start sites of genes that produce long-lived transcripts.

Self-transcribing active regulatory region sequencing (STARR-seq)

A method that assays enhancer activity for millions ofcandidate sequences by cloning them downstream of a reporter gene and upstream of a cleavage and polyadenylation site Functionally active enhancers drive expression of RNA molecules that contains the candidate sequence.

Degron system

A tool for rapidly degrading a specific protein in a cell. Genome editing is used to tag the protein of interest with a protein domain that is recognized by the E3 ubiquitin ligase complex. On addition of a small molecule, the tagged factor is inducibly degraded by the proteasome.

RNA aptamers

Structured RNA molecules selected for binding to a factor of interest to disrupt its functions or interactions.

Super-resolution microscopy

A collective term for light microscopy techniques that provide higher resolution (<200 nm) than imposed by the diffraction limit of visible light.

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Wissink, E.M., Vihervaara, A., Tippens, N.D. et al. Nascent RNA analyses: tracking transcription and its regulation. Nat Rev Genet 20, 705–723 (2019). https://doi.org/10.1038/s41576-019-0159-6

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