title: Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.
abstract: Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, hkimura@bio.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hayashi-Takanaka
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stasevich
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
mesh_terms: D000818:Animals; D044127:Epigenesis, Genetic / Q000235:genetics*; D006801:Humans; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011506:Proteins