title: Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.
abstract: Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.
authors.affiliation: Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907, USA pvidi@purdue.edu josephi@purdue.edu.
authors.forename: Pierre-Alexandre
authors.initials: PA
authors.lastname: Vidi
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907, USA Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Lelièvre
authors.affiliation: Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA pvidi@purdue.edu josephi@purdue.edu.
authors.forename: Joseph M K
authors.initials: JM
authors.lastname: Irudayaraj
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects* / Q000528:radiation effects*; D004260:DNA Repair; D049452:Green Fluorescent Proteins / Q000235:genetics; D006657:Histones / Q000235:genetics*; D006801:Humans; D011839:Radiation, Ionizing; D014466:Ultraviolet Rays
chemical_list: D002843:Chromatin; D006657:Histones; D001761:Bleomycin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate
keywords: Chromatin mobility; DNA damage; Paired-particle tracking