title: Quantifying histone and RNA polymerase II post-translational modification dynamics in mother and daughter cells.
abstract: Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; Dept. of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA; Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. Electronic address: tim.stasevich@colostate.edu.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Mab Institute Inc., Sapporo, Hokkaido 001-0021, Japan.
authors.forename: Naohito
authors.initials: N
authors.lastname: Nozaki
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan. Electronic address: hkimura@bio.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: D000107:Acetylation; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism
chemical_list: D006657:Histones; D007140:Immunoglobulin Fab Fragments; D012319:RNA Polymerase II
keywords: FabLEM; Gene regulation; Histone modifications; Live-cell imaging; RNA polymerase II