Flexible and dynamic nucleosome fiber in living mammalian cells

Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.

[1]  J. Dubochet,et al.  Cryo‐electron microscopy of vitrified chromosomes in situ. , 1986, The EMBO journal.

[2]  J. Langowski,et al.  How proteins squeeze through polymer networks: a Cartesian lattice study. , 2009, The Journal of chemical physics.

[3]  D. Bazett-Jones,et al.  Living without 30nm chromatin fibers. , 2011, Trends in biochemical sciences.

[4]  Elliot L. Elson,et al.  Fluorescence correlation spectroscopy : theory and applications , 2001 .

[5]  M. Tokunaga,et al.  Highly inclined thin illumination enables clear single-molecule imaging in cells , 2008, Nature Methods.

[6]  T. Cremer,et al.  Chromosome territories, nuclear architecture and gene regulation in mammalian cells , 2001, Nature Reviews Genetics.

[7]  K. Rippe,et al.  Genome organization: balancing stability and plasticity. , 2008, Biochimica et biophysica acta.

[8]  G. A. Blab,et al.  Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy. , 2001, Biophysical journal.

[9]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[10]  R. Kornberg.  Chromatin structure: a repeating unit of histones and DNA. , 1974, Science.

[11]  Jesse R. Dixon,et al.  Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions , 2012, Nature.

[12]  J. Sedat,et al.  A direct approach to the structure of eukaryotic chromosomes. , 1978, Cold Spring Harbor symposia on quantitative biology.

[13]  T. Cremer,et al.  Two-color fluorescence labeling of early and mid-to-late replicating chromatin in living cells , 2004, Chromosome Research.

[14]  Hiroshi Kimura,et al.  Kinetics of Core Histones in Living Human Cells , 2001, The Journal of cell biology.

[15]  J. Lippincott-Schwartz,et al.  Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. , 2009, Trends in cell biology.

[16]  Achilleas S Frangakis,et al.  Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ , 2008, Proceedings of the National Academy of Sciences.

[17]  C Cremer,et al.  Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. , 2000, Critical reviews in eukaryotic gene expression.

[18]  Hiroshi Kimura,et al.  Direct Imaging of DNA in Living Cells Reveals the Dynamics of Chromosome Formation , 1999, The Journal of cell biology.

[19]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[20]  Takeharu Nagai,et al.  Local nucleosome dynamics facilitate chromatin accessibility in living mammalian cells. , 2012, Cell reports.

[21]  Ugljesa Djuric,et al.  Open and closed domains in the mouse genome are configured as 10‐nm chromatin fibres , 2012, EMBO reports.

[22]  J. Langowski,et al.  Mapping eGFP Oligomer Mobility in Living Cell Nuclei , 2009, PloS one.

[23]  J. Daban.  High concentration of DNA in condensed chromatin. , 2003, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[24]  D. Agard,et al.  A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization , 1987, The Journal of cell biology.

[25]  Kazuhiro Maeshima,et al.  Chromatin structure: does the 30-nm fibre exist in vivo? , 2010, Current opinion in cell biology.

[26]  M. Tamura,et al.  Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs. , 2006, Biophysical journal.

[27]  Salman S Rogers,et al.  Precise particle tracking against a complicated background: polynomial fitting with Gaussian weight , 2007, Physical biology.

[28]  A Klug,et al.  Solenoidal model for superstructure in chromatin. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Gabriele Müller,et al.  Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. , 2003, Journal of molecular biology.

[30]  J. Langowski,et al.  Chromosome dynamics, molecular crowding, and diffusion in the interphase cell nucleus: a Monte Carlo lattice simulation study , 2010, Chromosome Research.

[31]  Gerald Kada,et al.  Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy , 2000, The EMBO journal.

[32]  J. Sedat,et al.  Spatial partitioning of the regulatory landscape of the X-inactivation centre , 2012, Nature.

[33]  H. Tanke,et al.  Chromatin movement visualized with photoactivable GFP-labeled histone H4. , 2008, Differentiation; research in biological diversity.

[34]  P. R. ten Wolde,et al.  Reaction Brownian dynamics and the effect of spatial fluctuations on the gain of a push-pull network. , 2008, The Journal of chemical physics.

[35]  K. Jaqaman,et al.  Robust single particle tracking in live cell time-lapse sequences , 2008, Nature Methods.

[36]  Jan Ellenberg,et al.  Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin , 2009, The EMBO journal.

[37]  A. Tanay,et al.  Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome , 2012, Cell.

[38]  C. Woodcock,et al.  Structural repeating units in chromatin. I. Evidence for their general occurrence. , 1976, Experimental cell research.

[39]  Grant J. Jensen,et al.  Chromatin in a marine picoeukaryote is a disordered assemblage of nucleosomes , 2013, Chromosoma.

[40]  P. Schwille,et al.  Fluorescence correlation spectroscopy in vivo , 2011 .

[41]  Raj Acharya,et al.  Spatio-temporal dynamics of genomic organization and function in the mammalian cell nucleus. , 2005, Advances in enzyme regulation.

[42]  N. Metropolis,et al.  Equation of State Calculations by Fast Computing Machines , 1953, Resonance.

[43]  D. E. Olins,et al.  Spheroid chromatin units (v bodies). , 1974, Science.

[44]  J. McNally,et al.  A benchmark for chromatin binding measurements in live cells , 2012, Nucleic acids research.

[45]  Yoshinori Nishino,et al.  Chromosomes without a 30-nm chromatin fiber , 2012, Nucleus.

[46]  J. B. Rattner,et al.  The higher-order structure of chromatin: evidence for a helical ribbon arrangement , 1984, The Journal of cell biology.

[47]  Karl Rohr,et al.  Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks , 2006, Chromosome Research.

[48]  Donald E. Olins,et al.  Spheroid Chromatin Units (ν Bodies) , 1974, Science.

[49]  Enrico Gratton,et al.  Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope. , 2005, Biophysical journal.

[50]  Achilleas S Frangakis,et al.  Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30‐nm chromatin structure , 2012, The EMBO journal.