Live Imaging of Transcription Sites Using an Elongating RNA Polymerase II-specific Probe

Overview

Journal J Cell Biol

Specialty Cell Biology

Date 2021 Dec 2

PMID 34854870

Citations 13

Authors

Satoshi Uchino

Yuma Ito

Yuko Sato

Tetsuya Handa

Yasuyuki Ohkawa

Makio Tokunaga

Hiroshi Kimura

Affiliations

Soon will be listed here.

Abstract

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.

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References

Kanno T, Kanno Y, LeRoy G, Campos E, Sun H, Brooks S . BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat Struct Mol Biol. 2014; 21(12):1047-57. PMC: 4720983. DOI: 10.1038/nsmb.2912. View

Suter D, Molina N, Gatfield D, Schneider K, Schibler U, Naef F . Mammalian genes are transcribed with widely different bursting kinetics. Science. 2011; 332(6028):472-4. DOI: 10.1126/science.1198817. View

Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y . Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells. Sci Adv. 2020; 6(25):eaaz6699. PMC: 7299619. DOI: 10.1126/sciadv.aaz6699. View

Hayashi-Takanaka Y, Yamagata K, Wakayama T, Stasevich T, Kainuma T, Tsurimoto T . Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res. 2011; 39(15):6475-88. PMC: 3159477. DOI: 10.1093/nar/gkr343. View

Nozaki T, Imai R, Tanbo M, Nagashima R, Tamura S, Tani T . Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging. Mol Cell. 2017; 67(2):282-293.e7. DOI: 10.1016/j.molcel.2017.06.018. View

Hou L, Wang Y, Liu Y, Zhang N, Shamovsky I, Nudler E . Paf1C regulates RNA polymerase II progression by modulating elongation rate. Proc Natl Acad Sci U S A. 2019; 116(29):14583-14592. PMC: 6642404. DOI: 10.1073/pnas.1904324116. View

Jackson D, Iborra F, Manders E, Cook P . Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol Biol Cell. 1998; 9(6):1523-36. PMC: 25378. DOI: 10.1091/mbc.9.6.1523. View

Manders E, Kimura H, Cook P . Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. J Cell Biol. 1999; 144(5):813-21. PMC: 2148202. DOI: 10.1083/jcb.144.5.813. View

Medlin J, Scurry A, Taylor A, Zhang F, Peterlin B, Murphy S . P-TEFb is not an essential elongation factor for the intronless human U2 snRNA and histone H2b genes. EMBO J. 2005; 24(23):4154-65. PMC: 1356315. DOI: 10.1038/sj.emboj.7600876. View

10.

Li J, Hsu A, Hua Y, Wang G, Cheng L, Ochiai H . Single-gene imaging links genome topology, promoter-enhancer communication and transcription control. Nat Struct Mol Biol. 2020; 27(11):1032-1040. PMC: 7644657. DOI: 10.1038/s41594-020-0493-6. View

11.

Guo Y, Manteiga J, Henninger J, Sabari B, DallAgnese A, Hannett N . Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature. 2019; 572(7770):543-548. PMC: 6706314. DOI: 10.1038/s41586-019-1464-0. View

12.

Ochiai H, Sugawara T, Yamamoto T . Simultaneous live imaging of the transcription and nuclear position of specific genes. Nucleic Acids Res. 2015; 43(19):e127. PMC: 4627063. DOI: 10.1093/nar/gkv624. View

13.

Sato Y, Kujirai T, Arai R, Asakawa H, Ohtsuki C, Horikoshi N . A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation. J Mol Biol. 2016; 428(20):3885-3902. DOI: 10.1016/j.jmb.2016.08.010. View

14.

Schoenfelder S, Sexton T, Chakalova L, Cope N, Horton A, Andrews S . Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet. 2009; 42(1):53-61. PMC: 3237402. DOI: 10.1038/ng.496. View

15.

Babokhov M, Hibino K, Itoh Y, Maeshima K . Local Chromatin Motion and Transcription. J Mol Biol. 2019; 432(3):694-700. DOI: 10.1016/j.jmb.2019.10.018. View

16.

Landgraf D, Okumus B, Chien P, Baker T, Paulsson J . Segregation of molecules at cell division reveals native protein localization. Nat Methods. 2012; 9(5):480-2. PMC: 3779060. DOI: 10.1038/nmeth.1955. View

17.

Pedelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G . Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2005; 24(1):79-88. DOI: 10.1038/nbt1172. View

18.

Tokunaga M, Imamoto N, Sakata-Sogawa K . Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods. 2008; 5(2):159-61. DOI: 10.1038/nmeth1171. View

19.

Tellier M, Zaborowska J, Caizzi L, Mohammad E, Velychko T, Schwalb B . CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation. Nucleic Acids Res. 2020; 48(14):7712-7727. PMC: 7641311. DOI: 10.1093/nar/gkaa514. View

20.

Parsons G, Spencer C . Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes. Mol Cell Biol. 1997; 17(10):5791-802. PMC: 232427. DOI: 10.1128/MCB.17.10.5791. View