Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters
Open accessAbstract
Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.
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Transcription factor binding to DNA appears to be very transient.
“While it is clear that enhancers typically favour the more flexible models of sequence organisation, the degree of necessary cooperation between TFs is less obvious, particularly since TF binding to DNA appears to be very transient [113–117].”
From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [116]
In Results, ‘The TATA Box Stabilizes Binding of the PIC’ (page unavailable), the paper says PIC formation at TATA-less promoters may be transient. It does not establish that transcription-factor binding to DNA is generally very transient; it instead reports more frequent PIC binding at TATA-containing promoters and notes that some general transcription factors may form a stable scaffold. (LLM, conf=0.87)
Experimental — accuracy not yet measuredSingle molecule footprinting can detect the binding of multiple transcription factors on a single DNA molecule.
“First developed in Drosophila, SMF can detect the binding of multiple TFs on a single DNA molecule [116].”
From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [116]
In Results, “Simultaneous Quantification of Multiple Transcription Initiation Intermediates” (page unavailable), the paper reports measuring multiple promoter-occupancy events on single molecules. It identifies footprints associated with the pre-initiation complex and RNA polymerase II, but does not show detection of multiple individual transcription factors on one molecule. The “Principles of Dual-Enzyme Single-Molecule Footprinting” section says the method was applied in Drosophila, not that SMF was first developed there. (LLM, conf=0.85)
Experimental — accuracy not yet measuredMolecular processes that transmit regulatory information from enhancers to promoters, and those associated with promoter operation itself, are stochastic and dynamic.
“Molecular processes that are thought to transmit regulatory information from enhancers to promoters (such as recruitment of transcription factors and coactivators, assembly of the Mediator complex40), as well as those that are associated with promoter operation itself (such as pre-initiation complex assembly, RNA polymerase II pausing and release41,42) are also stochastic and dynamic43.”
From Nonlinear control of transcription through enhancer–promoter interactions (2022) · cites this paper as [42]
The Discussion reports continuous, rapid RNA polymerase II turnover at paused promoters, supporting the claim that promoter operation is dynamic. The paper does not establish that enhancer-to-promoter processes, such as transcription-factor and coactivator recruitment or Mediator assembly, are stochastic and dynamic. No page numbers are available in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measuredIn many tractable experimental systems, increasing the number of TF binding sites leads to strongly nonlinear effects on gene expression.
“in many tractable experimental systems, increasing the number of sites leads to strongly nonlinear effects on gene expression2,3”
From Single-molecule states link transcription factor binding to gene expression (2024) · cites this paper as [3]
The Results section, “Simultaneous Quantification of Multiple Transcription Initiation Intermediates” (page unavailable), relates promoter activity to Pol II occupancy, not the number of transcription-factor binding sites. Searches for increasing binding-site number and nonlinear expression effects found no passage addressing that relationship in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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Cites
Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE
Roy, S., Ernst, J., Kharchenko, P. V., Kheradpour, P., Negre, N. N., Eaton, M. L., Landolin, J. M., Bristow, C. A., Ma, L., Lin, M. F., et al. (2010) · Science · doi:10.1126/science.1198374 · Cited by 1,223 · see citing works
Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq
Arnold, C. D., Gerlach, D., Stelzer, C., Boryń, Ł. M., Rath, M., & Stark, A. (2013) · Science · doi:10.1126/science.1232542 · Cited by 1,125 · see citing works
Getting up to speed with transcription elongation by RNA polymerase II
Jonkers, I., & Lis, J. T. (2015) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm3953 · Cited by 876 · see citing works
Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing
Kwak, H., Fuda, N. J., Core, L. J., & Lis, J. T. (2013) · Science · doi:10.1126/science.1229386 · Cited by 849 · see citing works
Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules
Kelly, T. K., Liu, Y., Lay, F. D., Liang, G., Berman, B. P., & Jones, P. A. (2012) · Genome Research · doi:10.1101/gr.143008.112 · Cited by 455 · see citing works
A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila
Saito, K., Inagaki, S., Mitsuyama, T., Kawamura, Y., Ono, Y., Sakota, E., Kotani, H., Asai, K., Siomi, H., & Siomi, M. C. (2009) · Nature · doi:10.1038/nature08501 · Cited by 440 · see citing works
Nechaev, S., Fargo, D. C., dos Santos, G., Liu, L., Gao, Y., & Adelman, K. (2010) · Science · doi:10.1126/science.1181421 · Cited by 424 · see citing works
A transcription reinitiation intermediate that is stabilized by activator
Yudkovsky, N., Ranish, J. A., & Hahn, S. (2000) · Nature · doi:10.1038/35041603 · Cited by 421 · see citing works
Gilchrist, D. A., dos Santos, G., Fargo, D. C., Xie, B., Gao, Y., Li, L., & Adelman, K. (2010) · Cell · doi:10.1016/j.cell.2010.10.004 · Cited by 414 · see citing works
QuasR: quantification and annotation of short reads in R
Gaidatzis, D., Lerch, A., Hahne, F., & Stadler, M. B. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu781 · Cited by 316 · see citing works
Self-citationP-TEFb kinase recruitment and function at heat shock lociPreprint
Lis, J. T., Mason, P., Peng, J., Price, D. H., & Werner, J. K. (2000) · Genes & Development · doi:10.1101/gad.14.7.792 · Cited by 314 · see citing works
Paused RNA Polymerase II as a Developmental Checkpoint
Levine, M. (2011) · Cell · doi:10.1016/j.cell.2011.04.021 · Cited by 288 · see citing works
Defining the Status of RNA Polymerase at Promoters
Core, L. J., Waterfall, J. J., Gilchrist, D. A., Fargo, D. C., Kwak, H., Adelman, K., & Lis, J. T. (2012) · Cell Reports · doi:10.1016/j.celrep.2012.08.034 · Cited by 252 · see citing works
Paused RNA polymerase II inhibits new transcriptional initiation
Shao, W.-Q., & Zeitlinger, J. (2017) · Nature Genetics · doi:10.1038/ng.3867 · Cited by 240 · see citing works
Henriques, T., Gilchrist, D. A., Nechaev, S., Bern, M., Muse, G. W., Burkholder, A., Fargo, D. C., & Adelman, K. (2013) · Molecular Cell · doi:10.1016/j.molcel.2013.10.001 · Cited by 240 · see citing works
Structure of promoter-bound TFIID and model of human pre-initiation complex assembly
Louder, R. K., He, Y., López‐Blanco, J. R., Fang, J., Chacón, P., & Nogales, E. (2016) · Nature · doi:10.1038/nature17394 · Cited by 233 · see citing works
The MTE, a new core promoter element for transcription by RNA polymerase IIPreprint
Lim, C. Y., Santoso, B., Boulay, T., Dong, E., Ohler, U., & Kadonaga, J. T. (2004) · Genes & Development · doi:10.1101/gad.1193404 · Cited by 216 · see citing works
NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila
Lee, C., Li, X., Hechmer, A., Eisen, M. B., Biggin, M. D., Venters, B. J., Jiang, C., Li, J., Pugh, B. F., & Gilmour, D. S. (2008) · Molecular and Cellular Biology · doi:10.1128/mcb.02224-07 · Cited by 214 · see citing works
Brannan, K. W., Kim, H., Erickson, B., Glover-Cutter, K. M., Kim, S., Fong, N., Kiemele, L., Hansen, K. C., Davis, R. E., Lykke‐Andersen, J., et al. (2012) · Molecular Cell · doi:10.1016/j.molcel.2012.03.006 · Cited by 205 · see citing works
Wagschal, A., Rousset, E., Basavarajaiah, P., Contreras, X., Harwig, A., Laurent‐Chabalier, S., Nakamura, M., Chen, X., Zhang, K., Méziane, O., et al. (2012) · Cell · doi:10.1016/j.cell.2012.08.004 · Cited by 177 · see citing works
Wirbelauer, C., Bell, O., & Schübeler, D. (2005) · Genes & Development · doi:10.1101/gad.347705 · Cited by 175 · see citing works
Self-citationRNA polymerase II pausing during development
Gaertner, B., & Zeitlinger, J. (2014) · Development · doi:10.1242/dev.088492 · Cited by 128 · see citing works
Mechanistic Interpretation of Promoter-Proximal Peaks and RNAPII Density Maps
Ehrensberger, A. H., Kelly, G. P., & Svejstrup, J. Q. (2013) · Cell · doi:10.1016/j.cell.2013.07.032 · Cited by 125 · see citing works
Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State
Cianfrocco, M. A., Kassavetis, G. A., Grob, P., Fang, J., Juven‐Gershon, T., Kadonaga, J. T., & Nogales, E. (2013) · Cell · doi:10.1016/j.cell.2012.12.005 · Cited by 121 · see citing works
Liang, K., Woodfin, A. R., Slaughter, B. D., Unruh, J. R., Box, A., Rickels, R. A., Gao, X., Haug, J. S., Jaspersen, S. L., & Shilatifard, A. (2015) · Molecular Cell · doi:10.1016/j.molcel.2015.09.021 · Cited by 118 · see citing works
Bell, O., Schwaiger, M., Oakeley, E. J., Lienert, F., Beisel, C., Stadler, M. B., & Schübeler, D. (2010) · Nature Structural & Molecular Biology · doi:10.1038/nsmb.1825 · Cited by 110 · see citing works
Self-citationDrosophila Heat Shock System as a General Model to Investigate Transcriptional RegulationPreprint
Guertin, M. J., Petesch, S. J., Zobeck, K. L., Min, I. M., & Lis, J. T. (2010) · Cold Spring Harbor Symposia on Quantitative Biology · doi:10.1101/sqb.2010.75.039 · Cited by 76 · see citing works
Hu, D., Smith, E. R., Garruss, A. S., Mohaghegh, N., Varberg, J. M., Lin, C., Jackson, J., Gao, X., Saraf, A. P., Florens, L. A., et al. (2013) · Molecular Cell · doi:10.1016/j.molcel.2013.07.003 · Cited by 72 · see citing works
Gal‐Yam, E. N., Jeong, S., Tanay, A., Egger, G., Lee, A. S., & Jones, P. A. (2006) · PLoS Genetics · doi:10.1371/journal.pgen.0020160 · Cited by 70 · see citing works
Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulationPreprint
Buckley, M. S., Kwak, H., Zipfel, W. R., & Lis, J. T. (2014) · Genes & Development · doi:10.1101/gad.231886.113 · Cited by 58 · see citing works
Nabilsi, N. H., Deleyrolle, L. P., Darst, R. P., Riva, A., Reynolds, B. A., & Kladde, M. P. (2013) · Genome Research · doi:10.1101/gr.161737.113 · Cited by 53 · see citing works
Genomic Organization of Human Transcription Initiation Complexes
Pugh, B. F., & Venters, B. J. (2016) · PLoS ONE · doi:10.1371/journal.pone.0149339 · Cited by 51 · see citing works
Levo, M., Avnit-Sagi, T., Lotan-Pompan, M., Kalma, Y., Weinberger, A., Yakhini, Z., & Segal, E. (2017) · Molecules and Cells · doi:10.1016/j.molcel.2017.01.007 · Cited by 49 · see citing works
Flagged first, then by citations
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Chromatin accessibility and the regulatory epigenome
Klemm, S. L., Shipony, Z., & Greenleaf, W. J. (2019) · Nature Reviews Genetics · doi:10.1038/s41576-018-0089-8 · Cited by 1,793 · see citing works
Born to run: control of transcription elongation by RNA polymerase II
Chen, F. X., Smith, E. R., & Shilatifard, A. (2018) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-018-0010-5 · Cited by 447 · see citing works
Nonlinear control of transcription through enhancer–promoter interactions
Zuin, J., Roth, G., Zhan, Y., Cramard, J., Redolfi, J., Piskadlo, E., Mach, P., Kryzhanovska, M., Tihanyi, G., Kohler, H., et al. (2022) · Nature · doi:10.1038/s41586-022-04570-y · Cited by 415 · see citing works
Cohesin and CTCF control the dynamics of chromosome folding
Mach, P., Kos, P. I., Zhan, Y., Cramard, J., Gaudin, S., Tünnermann, J., Marchi, E., Eglinger, J., Zuin, J., Kryzhanovska, M., et al. (2022) · Nature Genetics · doi:10.1038/s41588-022-01232-7 · Cited by 269 · see citing works
Transcription in Living Cells: Molecular Mechanisms of Bursting
Rodriguez, J., & Larson, D. R. (2020) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-011520-105250 · Cited by 224 · see citing works
Huang, K.-L., Jee, D., Stein, C. B., Elrod, N. D., Henriques, T., Mascibroda, L. G., Baillat, D., Russell, W. K., Adelman, K., & Wagner, E. J. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.08.016 · Cited by 184 · see citing works
Long-range single-molecule mapping of chromatin accessibility in eukaryotes
Shipony, Z., Marinov, G. K., Swaffer, M. P., Sinnott-Armstrong, N. A., Skotheim, J. M., Kundaje, A., & Greenleaf, W. J. (2020) · Nature Methods · doi:10.1038/s41592-019-0730-2 · Cited by 160 · see citing works
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Lammers, N. C., Kim, Y. J., Zhao, J., & Garcia, H. G. (2020) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2020.08.001 · Cited by 99 · see citing works
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Ray-Jones, H., & Spivakov, M. (2021) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-021-03903-w · Cited by 81 · see citing works
Single-molecule states link transcription factor binding to gene expression
Doughty, B. R., Hinks, M. M., Schaepe, J. M., Marinov, G. K., Thurm, A. R., Rios-Martinez, C., Parks, B. E., Tan, Y., Marklund, E., Dubocanin, D., et al. (2024) · Nature · doi:10.1038/s41586-024-08219-w · Cited by 42 · see citing works
Organismal benefits of transcription speed control at gene boundaries
Leng, X., Ivanov, M., Kindgren, P., Malik, I., Thieffry, A., Brodersen, P., Sandelin, A., Kaplan, C. D., & Marquardt, S. (2020) · The EMBO Reports · doi:10.15252/embr.201949315 · Cited by 40 · see citing works
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- Authors
- Krebs, A. R., Imanci, D., Hoerner, L., Gaidatzis, D., Burger, L., & Schübeler, D.
- Affiliations
- Friedrich Miescher Institute (CH)
- SIB Swiss Institute of Bioinformatics (CH)
- University of Basel (CH)
- Corresponding author
- Krebs, Arnaud R.
- Schübeler, Dirk
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2017-08
- Volume
- 67
- Issue
- 3
- Pages
- 411-422.e4
- ISSN
- 1097-2765 (ISSN-L)
- PMC
- PMC5548954
- OpenAlex
- W2738668193
- Cited by
- 249
- References
- 33
- Citations by year
- 2026: 33 · 2025: 37 · 2024: 33 · 2023: 20 · 2022: 26 · 2021: 29 · 2020: 27 · 2019: 22 · 2018: 18 · 2017: 2 · 2013: 1
- FWCI
- 7.71
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Cell (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- Novartis Research Foundation
- European Union (NoE “EpiGeneSys”, FP7-257082)
- “Blueprint” consortium (FP7-282510)
- European Research Council (ERC (204264 “EpiGePlas”)
- Swiss National Science Foundation (31003A_156963)
- Swiss National Science Foundation Ambizione (PZOOP3_161493)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
- Keywords
- RNA polymerase II, transcriptional pausing, transcription initiation, promoter structure, nascent RNA, early termination, heat shock promoter, Drosophila, preinitiation complex
- MeSH terms
- Single Molecule Imaging, Animals, Binding Sites, DNA, Drosophila melanogaster, Half-Life, Kinetics, Promoter Regions, Genetic, Protein Binding, RNA, RNA Polymerase II, Transcription, Genetic, TATA Box, HSP70 Heat-Shock Proteins, Transcription Initiation Site, Drosophila Proteins, Genome-Wide Association Study, Protein Stability, Proteolysis, Transcription Initiation, Genetic, Transcription Termination, Genetic
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