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      Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II

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          Significance

          Transcription by RNA Polymerase II (Pol II) is a highly dynamic process that is tightly regulated at each step of the transcription cycle. We generated GFP-RPB1 knockin cells and developed photobleaching of endogenous Pol II combined with computational modeling to study the in vivo dynamics of Pol II in real time. This approach allowed us to dissect promoter-paused Pol II from initiating and elongating Pol II and showed that initiation and promoter proximal pausing are surprisingly dynamic events, due to premature termination of Pol II. Our study provides new insights into Pol II dynamics and suggests that the iterative release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

          Abstract

          Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell–imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

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          Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.

          Karen Adelman, John Lis (2012)
          Recent years have witnessed a sea change in our understanding of transcription regulation: whereas traditional models focused solely on the events that brought RNA polymerase II (Pol II) to a gene promoter to initiate RNA synthesis, emerging evidence points to the pausing of Pol II during early elongation as a widespread regulatory mechanism in higher eukaryotes. Current data indicate that pausing is particularly enriched at genes in signal-responsive pathways. Here the evidence for pausing of Pol II from recent high-throughput studies will be discussed, as well as the potential interconnected functions of promoter-proximally paused Pol II.
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            Getting up to speed with transcription elongation by RNA polymerase II.

            Iris Jonkers, John Lis (2015)
            Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.
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              Controlling the elongation phase of transcription with P-TEFb.

              B. Matija Peterlin, David Price (2006)
              The positive transcription elongation factor b (P-TEFb) is a cyclin-dependent kinase that controls the elongation phase of transcription by RNA polymerase II (RNAPII). This process is made possible by the reversal of effects of negative elongation factors that include NELF and DSIF. In complex organisms, elongation control is critical for the regulated expression of most genes. In those organisms, the function of P-TEFb is influenced negatively by HEXIM proteins and 7SK snRNA and positively by a variety of recruiting factors. Phylogenetic analyses of the components of the human elongation control machinery indicate that the number of mechanisms utilized to regulate P-TEFb function increased as organisms developed more complex developmental patterns.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Proc Natl Acad Sci U S A
                Journal ID (iso-abbrev): Proc. Natl. Acad. Sci. U.S.A
                Journal ID (hwp): pnas
                Journal ID (pmc): pnas
                Journal ID (publisher-id): PNAS
                Title: Proceedings of the National Academy of Sciences of the United States of America
                Publisher: National Academy of Sciences
                ISSN (Print): 0027-8424
                ISSN (Electronic): 1091-6490
                Publication date (Print): 8 May 2018
                Publication date (Electronic): 9 April 2018
                Publication date PMC-release: 9 April 2018
                Volume: 115
                Issue: 19
                Pages: E4368-E4376
                Affiliations
                [1] aDepartment of Molecular Genetics, Erasmus Medical Center , 3015 AA Rotterdam, The Netherlands;
                [2] bOncode Institute, Erasmus Medical Center , 3015 AA Rotterdam, The Netherlands;
                [3] cDepartment of Pathology, Optical Imaging Centre, Erasmus Medical Center , 3015 AA Rotterdam, The Netherlands
                Author notes
                1To whom correspondence should be addressed. Email: J.Marteijn@ 123456erasmusmc.nl .

                Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved March 12, 2018 (received for review October 17, 2017)

                Author contributions: J.A.M. designed research; B.S., R.C.J., and M.E.G. performed research; B.S., R.C.J., B.G., F.W., A.F.T., S.D., J.P., W.A.v.C., and A.B.H. contributed new reagents/analytic tools; B.S., R.C.J., B.G., M.E.G., J.C., S.D., J.P., W.A.v.C., A.B.H., and J.A.M. analyzed data; and B.S. and J.A.M. wrote the paper.

                Article
                Publisher ID: 201717920
                DOI: 10.1073/pnas.1717920115
                PMC ID: 5948963
                PubMed ID: 29632207
                SO-VID: 4a19d033-141c-4b77-a411-d03e4d1baab6
                Copyright © Copyright © 2018 the Author(s). Published by PNAS.
                License:

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 9
                Funding
                Funded by: KWF Kankerbestrijding (Dutch Cancer Society) 501100004622
                Award ID: 10506/2016-1
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) 501100003246
                Award ID: 912.12.132
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) 501100003246
                Award ID: 854.11.002
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) 501100003246
                Award ID: 935.11.042
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) 501100003246
                Award ID: 864.13.004
                Categories
                Subject: PNAS Plus
                Subject: Biological Sciences
                Subject: Cell Biology
                Series title: From the Cover
                Series title: PNAS Plus

                Keywords: rna polymerase ii,transcription,transcription dynamics,promoter-proximal pausing,live-cell imaging
                Data availability:
                Keywords: rna polymerase ii, transcription, transcription dynamics, promoter-proximal pausing, live-cell imaging

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