title: A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.
abstract: Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.
authors.affiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Electronic address: satoy@bio.titech.ac.jp.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kujirai
authors.affiliation: Cell Architecture Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Arai
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Asakawa
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan.
authors.forename: Chizuru
authors.initials: C
authors.lastname: Ohtsuki
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Horikoshi
authors.affiliation: Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa City, Wakayama 649-6493, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamagata
authors.affiliation: Center for Education in Laboratory Animal Research, Chubu University, Kasugai, Aichi 487-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ueda
authors.affiliation: Public Relations Team, Kazusa DNA Research Institute, Chiba 292-0818, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan; Advanced ICT Research Institute, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan.
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan; Advanced ICT Research Institute, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.affiliation: Cell Architecture Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Akatsuki
authors.initials: A
authors.lastname: Kimura
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
authors.affiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Electronic address: hkimura@bio.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D015870:Gene Expression; D017930:Genes, Reporter; D006657:Histones / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008745:Methylation; D051379:Mice; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D057127:Single-Chain Antibodies / Q000032:analysis / Q000737:chemistry / Q000235:genetics
chemical_list: D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: Histone modification; Inactive X chromosome; Intracellular antibody; Live-cell imaging