title: Regulation of RNA polymerase II activation by histone acetylation in single living cells.
abstract: In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA [3] Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama, 332-0012, Japan [3] Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hayashi-Takanaka
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Advanced Medical Initiatives, Faculty of Medicine, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Maehara
authors.affiliation: 1] Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama, 332-0012, Japan [2] Department of Advanced Medical Initiatives, Faculty of Medicine, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Ohkawa
authors.affiliation: 1] Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan [2] RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, 230-0045, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Sakata-Sogawa
authors.affiliation: 1] Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan [2] RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, 230-0045, Japan.
authors.forename: Makio
authors.initials: M
authors.lastname: Tokunaga
authors.affiliation: Department of Biotechnology Research, Kazusa DNA Research Institute, Chiba, 292-0818, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
authors.affiliation: Mab Institute Inc., Sapporo, 001-0021, Japan.
authors.forename: Naohito
authors.initials: N
authors.lastname: Nozaki
authors.affiliation: 1] Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA [2] Institute for Soft Matter and Functional Materials, Helmholtz Zentrum Berlin, Berlin, 14109, Germany.
authors.forename: James G
authors.initials: JG
authors.lastname: McNally
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama, 332-0012, Japan [3] Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: D000107:Acetylation; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D047369:Chromatin Immunoprecipitation; D004789:Enzyme Activation; D016678:Genome / Q000235:genetics; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000378:metabolism*; D059010:Single-Cell Analysis; D013997:Time Factors; D061805:Transcription Elongation, Genetic; D061785:Transcription Initiation, Genetic; D014158:Transcription, Genetic
chemical_list: D006657:Histones; D012319:RNA Polymerase II; D008239:Lysine