title: Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.
abstract: Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hayashi-Takanaka
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamagata
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Wakayama
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.forename: Takashi
authors.initials: T
authors.lastname: Kainuma
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tsurimoto
authors.forename: Makoto
authors.initials: M
authors.lastname: Tachibana
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Shinkai
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
authors.forename: Naohito
authors.initials: N
authors.lastname: Nozaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001755:Blastocyst / Q000378:metabolism; D002478:Cells, Cultured; D041321:Chromosomes, Human, X; D044127:Epigenesis, Genetic; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments; D008239:Lysine / Q000378:metabolism; D008297:Male; D008745:Methylation / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D059010:Single-Cell Analysis
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D007140:Immunoglobulin Fab Fragments; C012589:trichostatin A; D008239:Lysine