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Sample records for 30-nm chromatin fibers

  1. Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Holley, W. R.; Mian, I. S.; Chatterjee, A.

    1998-01-01

    A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Copyright 1998 Academic Press.

  2. Implicit Solvent Model for Million-Atom Atomistic Simulations: Insights into the Organization of 30-nm Chromatin Fiber.

    PubMed

    Izadi, Saeed; Anandakrishnan, Ramu; Onufriev, Alexey V

    2016-12-13

    Molecular dynamics (MD) simulations based on the implicit solvent generalized Born (GB) models can provide significant computational advantages over the traditional explicit solvent simulations. However, the standard GB becomes prohibitively expensive for all-atom simulations of large structures; the model scales poorly, ∼n(2), with the number of solute atoms. Here we combine our recently developed optimal point charge approximation (OPCA) with the hierarchical charge partitioning (HCP) approximation to present an ∼n log n multiscale, yet fully atomistic, GB model (GB-HCPO). The HCP approximation exploits the natural organization of biomolecules (atoms, groups, chains, and complexes) to partition the structure into multiple hierarchical levels of components. OPCA approximates the charge distribution for each of these components by a small number of point charges so that the low order multipole moments of these components are optimally reproduced. The approximate charges are then used for computing electrostatic interactions with distant components, while the full set of atomic charges are used for nearby components. We show that GB-HCPO can deliver up to 2 orders of magnitude speedup compared to the standard GB, with minimal impact on its accuracy. For large structures, GB-HCPO can approach the same nominal speed, as in nanoseconds per day, as the highly optimized explicit-solvent simulation based on particle mesh Ewald (PME). The increase in the nominal simulation speed, relative to the standard GB, coupled with substantially faster sampling of conformational space, relative to the explicit solvent, makes GB-HCPO a suitable candidate for MD simulation of large atomistic systems in implicit solvent. As a practical demonstration, we use GB-HCPO simulation to refine a ∼1.16 million atom structure of 30 nm chromatin fiber (40 nucleosomes). The refined structure suggests important details about spatial organization of the linker DNA and the histone tails in the

  3. Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure

    PubMed Central

    Bajpai, Gaurav; Jain, Ishutesh; Inamdar, Mandar M.; Das, Dibyendu; Padinhateeri, Ranjith

    2017-01-01

    Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions. PMID:28135276

  4. Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

    PubMed Central

    Nishino, Yoshinori; Eltsov, Mikhail; Joti, Yasumasa; Ito, Kazuki; Takata, Hideaki; Takahashi, Yukio; Hihara, Saera; Frangakis, Achilleas S; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, Kazuhiro

    2012-01-01

    How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures. PMID:22343941

  5. The shades of gray of the chromatin fiber: recent literature provides new insights into the structure of chromatin.

    PubMed

    Ausió, Juan

    2015-01-01

    The chromatin fiber consists of a string of nucleosomes connected by linker DNA regions. The hierarchy of folding of this fiber within the cell has long been controversial, and the existence of an originally described 30 nm fiber has been debated and reviewed extensively. This review contextualizes two recent papers on this topic that suggest the 30 nm fiber to be an over-simplification. The idealized model from the first study provides good insight into the constraints and histone participation in the maintenance of the fiber structure. The second paper provides a theoretical description of a more realistic view of the highly heterogeneous and dynamic chromatin organization in the in vivo setting. It is now time to abandon the highly regular "one start" solenoidal 30 nm structure and replace it with a more realistic highly dynamic, polymorphic fiber.

  6. Chromatin fiber polymorphism triggered by variations of DNA linker lengths.

    PubMed

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-06-03

    Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatin's diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes.

  7. Chromatin fiber functional organization: Some plausible models

    NASA Astrophysics Data System (ADS)

    Lesne, A.; Victor, J.-M.

    2006-03-01

    We here present a modeling study of the chromatin fiber functional organization. Multi-scale modeling is required to unravel the complex interplay between the fiber and the DNA levels. It suggests plausible scenarios, including both physical and biological aspects, for fiber condensation, its targeted decompaction, and transcription regulation. We conclude that a major role of the chromatin fiber structure might be to endow DNA with allosteric potentialities and to control DNA transactions by an epigenetic tuning of its mechanical and topological constraints.

  8. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    NASA Astrophysics Data System (ADS)

    Lesne, Annick; Bécavin, Christophe; Victor, Jean–Marc

    2012-02-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

  9. Nucleosome interactions in chromatin: Fiber stiffening and hairpin formation

    NASA Astrophysics Data System (ADS)

    Mergell, Boris; Everaers, Ralf; Schiessel, Helmut

    2004-07-01

    We use Monte Carlo simulations to study attractive and excluded volume interactions between nucleosome core particles in 30-nm chromatin fibers. The nucleosomes are treated as disklike objects having an excluded volume and short-range attraction modeled by a variant of the Gay-Berne potential. The nucleosomes are connected via bendable and twistable linker DNA in the crossed linker fashion. We investigate the influence of the nucleosomal excluded volume on the stiffness of the fiber. For parameter values that correspond to chicken erythrocyte chromatin, we find that the persistence length is governed to a large extent by that excluded volume whereas the soft linker backbone elasticity plays only a minor role. We further find that internucleosomal attraction can induce the formation of hairpin configurations. Tension-induced opening of such configurations into straight fibers manifests itself in a quasiplateau in the force-extension curve that resembles results from recent micromanipulation experiments. Such hairpins may play a role in the formation of higher-order structures in chromosomes like chromonema fibers.

  10. Chromatin Fiber Dynamics under Tension and Torsion

    PubMed Central

    Lavelle, Christophe; Victor, Jean-Marc; Zlatanova, Jordanka

    2010-01-01

    Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism. PMID:20480035

  11. Changing chromatin fiber conformation by nucleosome repositioning.

    PubMed

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-11-04

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.

  12. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-01-01

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell. PMID:25418099

  13. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

    Boulé, Jean-Baptiste; Mozziconacci, Julien; Lavelle, Christophe

    2015-01-01

    In eukaryotes, the genome is packed into chromosomes, each consisting of large polymeric fibers made of DNA bound with proteins (mainly histones) and RNA molecules. The nature and precise 3D organization of this fiber has been a matter of intense speculations and debates. In the emerging picture, the local chromatin state plays a critical role in all fundamental DNA transactions, such as transcriptional control, DNA replication or repair. However, the molecular and structural mechanisms involved remain elusive. The purpose of this review is to give an overview of the tremendous efforts that have been made for almost 40 years to build physiologically relevant models of chromatin structure. The motivation behind building such models was to shift our representation and understanding of DNA transactions from a too simplistic ‘naked DNA’ view to a more realistic ‘coated DNA’ view, as a step towards a better framework in which to interpret mechanistically the control of genetic expression and other DNA metabolic processes. The field has evolved from a speculative point of view towards in vitro biochemistry and in silico modeling, but is still longing for experimental in vivo validations of the proposed structures or even proof of concept experiments demonstrating a clear role of a given structure in a metabolic transaction. The mere existence of a chromatin fiber as a relevant biological entity in vivo has been put into serious questioning. Current research is suggesting a possible reconciliation between theoretical studies and experiments, pointing towards a view where the polymorphic and dynamic nature of the chromatin fiber is essential to support its function in genome metabolism.

  14. Chromatin fiber allostery and the epigenetic code

    NASA Astrophysics Data System (ADS)

    Lesne, Annick; Foray, Nicolas; Cathala, Guy; Forné, Thierry; Wong, Hua; Victor, Jean-Marc

    2015-02-01

    The notion of allostery introduced for proteins about fifty years ago has been extended since then to DNA allostery, where a locally triggered DNA structural transition remotely controls other DNA-binding events. We further extend this notion and propose that chromatin fiber allosteric transitions, induced by histone-tail covalent modifications, may play a key role in transcriptional regulation. We present an integrated scenario articulating allosteric mechanisms at different scales: allosteric transitions of the condensed chromatin fiber induced by histone-tail acetylation modify the mechanical constraints experienced by the embedded DNA, thus possibly controlling DNA-binding of allosteric transcription factors or further allosteric mechanisms at the linker DNA level. At a higher scale, different epigenetic constraints delineate different statistically dominant subsets of accessible chromatin fiber conformations, which each favors the assembly of dedicated regulatory complexes, as detailed on the emblematic example of the mouse Igf2-H19 gene locus and its parental imprinting. This physical view offers a mechanistic and spatially structured explanation of the observed correlation between transcriptional activity and histone modifications. The evolutionary origin of allosteric control supports to speak of an ‘epigenetic code’, by which events involved in transcriptional regulation are encoded in histone modifications in a context-dependent way.

  15. Computer simulation of the 30-nanometer chromatin fiber.

    PubMed Central

    Wedemann, Gero; Langowski, Jörg

    2002-01-01

    A new Monte Carlo model for the structure of chromatin is presented here. Based on our previous work on superhelical DNA and polynucleosomes, it reintegrates aspects of the "solenoid" and the "zig-zag" models. The DNA is modeled as a flexible elastic polymer chain, consisting of segments connected by elastic bending, torsional, and stretching springs. The electrostatic interaction between the DNA segments is described by the Debye-Hückel approximation. Nucleosome core particles are represented by oblate ellipsoids; their interaction potential has been parameterized by a comparison with data from liquid crystals of nucleosome solutions. DNA and chromatosomes are linked either at the surface of the chromatosome or through a rigid nucleosome stem. Equilibrium ensembles of 100-nucleosome chains at physiological ionic strength were generated by a Metropolis-Monte Carlo algorithm. For a DNA linked at the nucleosome stem and a nucleosome repeat of 200 bp, the simulated fiber diameter of 32 nm and the mass density of 6.1 nucleosomes per 11 nm fiber length are in excellent agreement with experimental values from the literature. The experimental value of the inclination of DNA and nucleosomes to the fiber axis could also be reproduced. Whereas the linker DNA connects chromatosomes on opposite sides of the fiber, the overall packing of the nucleosomes leads to a helical aspect of the structure. The persistence length of the simulated fibers is 265 nm. For more random fibers where the tilt angles between two nucleosomes are chosen according to a Gaussian distribution along the fiber, the persistence length decreases to 30 nm with increasing width of the distribution, whereas the other observable parameters such as the mass density remain unchanged. Polynucleosomes with repeat lengths of 212 bp also form fibers with the expected experimental properties. Systems with larger repeat length form fibers, but the mass density is significantly lower than the measured value. The

  16. Forced unraveling of chromatin fibers with nonuniform linker DNA lengths

    NASA Astrophysics Data System (ADS)

    Ozer, Gungor; Collepardo-Guevara, Rosana; Schlick, Tamar

    2015-02-01

    The chromatin fiber undergoes significant structural changes during the cell's life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous.

  17. Extended chromatin fibers: evidence from scanning force microscopy studies

    NASA Astrophysics Data System (ADS)

    Leuba, Sanford S.; Yang, Guoliang; Robert, Charles; van Holde, Kensal; Zlatanova, Jordanka; Bustamante, Carlos J.

    1995-03-01

    Unfixed chicken erythrocyte fibers in very low salt have been imaged using the scanning force microscope (SFM) operating in the tapping mode in air at ambient humidity. These images reveal a 3D organization of the fibers. The planar 'zig-zag' conformation is rare, and extended 'beads- on-a-string' fibers are seen only in chromatin depleted of H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies indicate that chromatin fibers may exist as irregular 3D arrays of nucleosomes even at low ionic strength. The basic subunit of chromatin, the nucleosome, is composed of a core particle of 146 bp of DNA wrapping 1.75 left-handed superhelical turns around an octamer of core histones and of DNA connecting consecutive core particles. The linker of lysine-rich histones (H1 family) bind the DNA entering and exiting the nucleosome core particle. We suggest that by binding the entry/exit DNA, histone H1 may fix the entry/exit DNA angle. The fixed entry/exit angle, the rigidity of the linker DNA at low ionic strength, and the natural variability of the linker DNA length determine an irregular 3D fiber of chromatin. Our results emphasize the role of H1 in determining the entry/exit DNA angle, which further helps determine the mutual disposition of adjacent nucleosomes an the packing of the chromatin fiber.

  18. The Chromatin Fiber: Multiscale Problems and Approaches

    PubMed Central

    Ozer, Gungor; Luque, Antoni; Schlick, Tamar

    2015-01-01

    The structure of chromatin, affected by many factors from DNA linker lengths to posttranslational modifications, is crucial to the regulation of eukaryotic cells. Combined experimental and computational methods have led to new insights into its structural and dynamical features, from interactions due to the flexible core histone tails of the nucleosomes to the physical mechanism driving the formation of chromosomal domains. Here we present a perspective of recent advances in chromatin modeling techniques at the atomic, mesoscopic, and chromosomal scales with a view toward developing multiscale computational strategies to integrate such findings. Innovative modeling methods that connect molecular to chromosomal scales are crucial for interpreting experiments and eventually deciphering the complex dynamic organization and function of chromatin in the cell. PMID:26057099

  19. Nucleosome geometry and internucleosomal interactions control the chromatin fiber conformation.

    PubMed

    Kepper, Nick; Foethke, Dietrich; Stehr, Rene; Wedemann, Gero; Rippe, Karsten

    2008-10-01

    Based on model structures with atomic resolution, a coarse-grained model for the nucleosome geometry was implemented. The dependence of the chromatin fiber conformation on the spatial orientation of nucleosomes and the path and length of the linker DNA was systematically explored by Monte Carlo simulations. Two fiber types were analyzed in detail that represent nucleosome chains without and with linker histones, respectively: two-start helices with crossed-linker DNA (CL conformation) and interdigitated one-start helices (ID conformation) with different nucleosome tilt angles. The CL conformation was derived from a tetranucleosome crystal structure that was extended into a fiber. At thermal equilibrium, the fiber shape persisted but relaxed into a structure with a somewhat lower linear mass density of 3.1 +/- 0.1 nucleosomes/11 nm fiber. Stable ID fibers required local nucleosome tilt angles between 40 degrees and 60 degrees. For these configurations, much higher mass densities of up to 7.9 +/- 0.2 nucleosomes/11 nm fiber were obtained. A model is proposed, in which the transition between a CL and ID fiber is mediated by relatively small changes of the local nucleosome geometry. These were found to be in very good agreement with changes induced by linker histone H1 binding as predicted from the high resolution model structures.

  20. Spontaneous access to DNA target sites in folded chromatin fibers.

    PubMed

    Poirier, Michael G; Bussiek, Malte; Langowski, Jörg; Widom, Jonathan

    2008-06-13

    DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from approximately 3-fold decreases to approximately 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as approximately 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.

  1. What Determines the Folding of the Chromatin Fiber?

    NASA Astrophysics Data System (ADS)

    van Holde, Kensal; Zlatanova, Jordanka

    1996-10-01

    In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels. We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription. We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms. On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence.

  2. Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

    PubMed

    Deng, Xiang; Zhironkina, Oxana A; Cherepanynets, Varvara D; Strelkova, Olga S; Kireev, Igor I; Belmont, Andrew S

    2016-09-26

    In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication.

  3. Unraveling chromatin structure using magnetic tweezers

    NASA Astrophysics Data System (ADS)

    van Noort, John

    2010-03-01

    The compact, yet dynamic organization of chromatin plays an essential role in regulating gene expression. Although the static structure of chromatin fibers has been studied extensively, the controversy about the higher order folding remains. The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all DNA processes. To understand the relation between gene regulation and chromatin structure it is essential to uncover the mechanisms by which chromatin fibers fold and unfold. We used magnetic tweezers to probe the mechanical properties of individual nucleosomes and chromatin fibers consisting of a single, well-defined array of 25 nucleosomes. From these studies five major features appeared upon forced extension of chromatin fibers: the elastic stretching of chromatin's higher order structure, the breaking of internucleosomal contacts, unwrapping of the first turn of DNA, unwrapping of the second turn of DNA, and the dissociation of histone octamers. These events occur sequentially at the increasing force. Neighboring nucleosomes stabilize DNA folding into a nucleosome relative to isolated nucleosomes. When an array of nucleosomes is folded into a 30 nm fiber, representing the first level of chromatin condensation, the fiber stretched like a Hookian spring at forces up to 4 pN. Together with a nucleosome-nucleosome stacking energy of 14 kT this points to a solenoid as the underlying topology of the 30 nm fiber. Surprisingly, linker histones do not affect the length or stiffness of the fibers, but stabilize fiber folding up to forces of 7 pN. The stiffness of the folded chromatin fiber points at histone tails that mediate nucleosome stacking. Fibers with a nucleosome repeat length of 167 bp instead of 197 bp are significantly stiffer, consistent with a two-start helical arrangement. The extensive thermal breathing of the chromatin fiber that is a consequence of the observed high compliance provides a structural basis for understanding the

  4. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo.

    PubMed

    Ricci, Maria Aurelia; Manzo, Carlo; García-Parajo, María Filomena; Lakadamyali, Melike; Cosma, Maria Pia

    2015-03-12

    Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state.

  5. Chromatin structure revealed by X-ray scattering analysis and computational modeling.

    PubMed

    Maeshima, Kazuhiro; Imai, Ryosuke; Hikima, Takaaki; Joti, Yasumasa

    2014-12-01

    It remains unclear how the 2m of human genomic DNA is organized in each cell. The textbook model has long assumed that the 11-nm-diameter nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, is folded into a 30-nm chromatin fiber. One of the classical models assumes that the 30-nm chromatin fiber is further folded helically to form a larger fiber. Small-angle X-ray scattering (SAXS) is a powerful method for investigating the bulk structure of interphase chromatin and mitotic chromosomes. SAXS can detect periodic structures in biological materials in solution. In our SAXS results, no structural feature larger than 11 nm was detected. Combining this with a computational analysis of "in silico condensed chromatin" made it possible to understand more about the X-ray scattering profiles and suggested that the chromatin in interphase nuclei and mitotic chromosomes essentially consists of irregularly folded nucleosome fibers lacking the 30-nm chromatin structure. In this article, we describe the experimental details of our SAXS and modeling systems. We also discuss other methods for investigating the chromatin structure in cells.

  6. Intercalation and buckling instability of DNA linker within locked chromatin fiber

    NASA Astrophysics Data System (ADS)

    Victor, Jean-Marc; Ben-Haïm, Eli; Lesne, A.

    2002-12-01

    The chromatin fiber is a complex of DNA and specific proteins called histones forming the first structural level of organization of eukaryotic chromosomes. In tightly organized chromatin fibers, the short segments of naked DNA linking the nucleosomes are strongly end constrained. Longitudinal thermal fluctuations in these linkers allow intercalative mode of protein binding. We show that mechanical constraints generated in the first stage of the binding process induce linker DNA buckling; buckling in turn modifies the binding energies and activation barriers and creates a force of decondensation at the chromatin fiber level. The unique structure and properties of DNA thus yield a novel physical mechanism of buckling instability that might play a key role in the regulation of gene expression.

  7. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  8. Toward multiscale modeling of the chromatin fiber: a coarse grain model for DNA

    NASA Astrophysics Data System (ADS)

    Savelyev, Alexey; Papoian, Garegin

    2008-03-01

    In eukaryotic cells DNA is compacted a million-fold into a chromatin. Understanding the mechanism of chromatin folding is of great biological importance. All-atom Molecular Dynamics (MD) simulations could provide crucial insights into the electrostatic and structural mechanisms of chromatin folding. However, because of the enormous size of even short chromatin fiber segment and long folding time-scales, atomistic simulations are computationally impractical. Our long-term aim is to build an accurate coarse-grain (CG) model of the chromatin, derived systematically from all-atom simulations of its smaller parts. Here we report the development of the CG model for a linear DNA chain, playing the role of a linker DNA segment in the chromatin. We derived CG inter-DNA electrostatic potential from atomistic simulations with explicit solvent and mobile ions, instead of relying on the standard models of continuum electrostatics, which are inadequate at small intermolecular distances. In addition, we used the ideas of renormalization group theory to construct an optimization scheme for parameterizing the CG force field. This novel approach is designed to accurately reproduce correlations among various CG degrees of freedom. The implementation of these correlations was left as an open question in the prior studies of CG polymer models.

  9. Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

    NASA Astrophysics Data System (ADS)

    Bystricky, Kerstin; Heun, Patrick; Gehlen, Lutz; Langowski, Jörg; Gasser, Susan M.

    2004-11-01

    Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of 110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure. higher-order structure | 30-nm fiber | nucleosomes

  10. Monte Carlo simulation of chromatin stretching

    NASA Astrophysics Data System (ADS)

    Aumann, Frank; Lankas, Filip; Caudron, Maïwen; Langowski, Jörg

    2006-04-01

    We present Monte Carlo (MC) simulations of the stretching of a single 30nm chromatin fiber. The model approximates the DNA by a flexible polymer chain with Debye-Hückel electrostatics and uses a two-angle zigzag model for the geometry of the linker DNA connecting the nucleosomes. The latter are represented by flat disks interacting via an attractive Gay-Berne potential. Our results show that the stiffness of the chromatin fiber strongly depends on the linker DNA length. Furthermore, changing the twisting angle between nucleosomes from 90° to 130° increases the stiffness significantly. An increase in the opening angle from 22° to 34° leads to softer fibers for small linker lengths. We observe that fibers containing a linker histone at each nucleosome are stiffer compared to those without the linker histone. The simulated persistence lengths and elastic moduli agree with experimental data. Finally, we show that the chromatin fiber does not behave as an isotropic elastic rod, but its rigidity depends on the direction of deformation: Chromatin is much more resistant to stretching than to bending.

  11. Hard x-ray Zernike microscopy reaches 30 nm resolution.

    SciTech Connect

    Chen, Y.; Chen, T.; Yi, J.; Chu, Y.; Lee, W.-K.; Wang, C.; Kempson, I.; Hwu, Y.; Gajdosik, V.; Margaritondo, G.

    2011-03-30

    Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30?nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

  12. Hard x-ray Zernike Microscopy Reaches 30 nm Resolution

    SciTech Connect

    Chen, Y.T.; Chu, Y.; Chen, T-Y.; Yi, J.; Lee, W-K.; Wang, C-L.; Kempson, I. M.; Hwu, Y.; Gajdosik, V.; Margaritondo, G.

    2011-03-30

    Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

  13. A novel double patterning approach for 30nm dense holes

    NASA Astrophysics Data System (ADS)

    Hsu, Dennis Shu-Hao; Wang, Walter; Hsieh, Wei-Hsien; Huang, Chun-Yen; Wu, Wen-Bin; Shih, Chiang-Lin; Shih, Steven

    2011-04-01

    Double Patterning Technology (DPT) was commonly accepted as the major workhorse beyond water immersion lithography for sub-38nm half-pitch line patterning before the EUV production. For dense hole patterning, classical DPT employs self-aligned spacer deposition and uses the intersection of horizontal and vertical lines to define the desired hole patterns. However, the increase in manufacturing cost and process complexity is tremendous. Several innovative approaches have been proposed and experimented to address the manufacturing and technical challenges. A novel process of double patterned pillars combined image reverse will be proposed for the realization of low cost dense holes in 30nm node DRAM. The nature of pillar formation lithography provides much better optical contrast compared to the counterpart hole patterning with similar CD requirements. By the utilization of a reliable freezing process, double patterned pillars can be readily implemented. A novel image reverse process at the last stage defines the hole patterns with high fidelity. In this paper, several freezing processes for the construction of the double patterned pillars were tested and compared, and 30nm double patterning pillars were demonstrated successfully. A variety of different image reverse processes will be investigated and discussed for their pros and cons. An economic approach with the optimized lithography performance will be proposed for the application of 30nm DRAM node.

  14. Sub-30-nm defect removal on EUV substrates

    NASA Astrophysics Data System (ADS)

    Rastegar, Abbas; Eichenlaub, Sean; Kadaksham, Arun John; House, Matt; Cha, Brian; Yun, Henry

    2009-01-01

    Naturally occurring sub 30 nm defects on quartz and Low Thermal Expansion Material (LTEM) substrates were characterized by using Atomic Force Microscope(AFM). Our data indicates that a majority of defects on the incoming substrate are hard defects including large, flat particles with a height less than 5 nm, tiny particles with a size of 10 nm to 30 nm SEVD and pits with a depth of about 9 nm. All the soft particles added by handling with sizes of >50 nm can be removed with a single cleaning process. At least four cleaning cycles are required to remove all of the remaining embedded particles. However, after particle removal in their initial location a shallow pit remains. Based on detailed characterization of defect and surface by AFM, we propose that these hard particles are added during the glass polishing step and therefore it is important to revisit the glass Chemical Mechanical Polishing (CMP) processes and optimize them for defect reduction. A qualitative value for particle removal efficiency (PRE) of >99% was obtained for 20 nm Poly Styrene Latex Sphere (PSL) deposited particles on surface of glass.

  15. Insights into interphase large-scale chromatin structure from analysis of engineered chromosome regions.

    PubMed

    Belmont, A S; Hu, Y; Sinclair, P B; Wu, W; Bian, Q; Kireev, I

    2010-01-01

    How chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions that preserve large-scale chromatin structure. Our results confirm the existence of large-scale chromatin domains and fibers formed by the folding of 10-nm and 30-nm chromatin fibers into larger, spatially distinct domains. Transcription at levels within severalfold of the levels measured for endogenous loci occur within these large-scale chromatin structures on a condensed template linearly compacted several hundred fold to 1000-fold relative to B-form DNA. However, transcriptional induction is accompanied by a severalfold decondensation of this large-scale chromatin structure that propagates hundreds of kilobases beyond the induced gene. Examination of engineered chromosome regions in mouse embryonic stem cells (ESCs) and differentiated cells suggests a surprising degree of plasticity in this large-scale chromatin structure, allowing long-range DNA interactions within the context of large-scale chromatin fibers. Recapitulation of gene-specific differences in large-scale chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.

  16. Insights into interphase large-scale chromatin structure from analysis of engineered chromosome regions

    PubMed Central

    Belmont, Andrew S.; Hu, Yan; Sinclair, Paul; Wu, Wei; Bian, Qian; Kireev, Igor

    2012-01-01

    How chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions which preserve large-scale chromatin structure. Our results confirm the existence of large-scale chromatin domains and fibers formed by the folding of 10 and 30 nm chromatin fibers into larger, spatially distinct domains. Transcription at levels within several fold of the levels measured for endogenous loci occur within these large-scale chromatin structures on a condensed template linearly compacted several hundred fold to one thousand fold relative to B-form DNA. However, transcriptional induction is accompanied by a several fold decondensation of this large-scale chromatin structure that propagates hundreds of kb beyond the induced gene. Examination of engineered chromosome regions in mouse ES and differentiated cells suggests a surprising degree of plasticity in this large-scale chromatin structure, allowing long-range DNA interactions within the context of large-scale chromatin fibers. Recapitulation of gene specific differences in large-scale chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture. PMID:21467143

  17. Incoherent magnetization reversal in 30-nm Ni particles

    NASA Astrophysics Data System (ADS)

    Ross, C. A.; Chantrell, R.; Hwang, M.; Farhoud, M.; Savas, T. A.; Hao, Y.; Smith, Henry I.; Ross, F. M.; Redjdal, M.; Humphrey, F. B.

    2000-12-01

    The magnetic properties of a 100-nm-period large-area array of regular, 30-nm polycrystalline nickel particles have been studied. The particles are found to reverse incoherently, and their hysteresis behavior has been compared with a computational model over a range of temperatures. Excellent agreement with the model is obtained, indicating that switching of the particles is dominated by the reversal of approximately 10-nm-diameter volumes within each particle. These switching volumes are identified with the columnar grains in the polycrystalline nickel, showing that the microstructure determines the magnetic behavior of the particles. This explains the anisotropy distribution and the onset of superparamagnetism in the sample. Incoherent reversal occurs even though the particles are only 1.5 times the exchange length in nickel, a size at which nearly uniform rotation is expected to occur if the particles were homogeneous.

  18. Chromatin hydrodynamics.

    PubMed

    Bruinsma, Robijn; Grosberg, Alexander Y; Rabin, Yitzhak; Zidovska, Alexandra

    2014-05-06

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.

  19. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.

  20. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    PubMed Central

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-01

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  1. Kilobase Pair Chromatin Fiber Contacts Promoted by Living-System-Like DNA Linker Length Distributions and Nucleosome Depletion.

    PubMed

    Bascom, Gavin D; Kim, Taejin; Schlick, Tamar

    2017-03-31

    Nucleosome placement, or DNA linker length patterns, are believed to yield specific spatial features in chromatin fibers, but details are unknown. Here we examine by mesoscale modeling how kilobase (kb) range contacts and fiber looping depend on linker lengths ranging from 18 to 45 bp, with values modeled after living systems, including nucleosome free regions (NFRs) and gene encoding segments. We also compare artificial constructs with alternating versus randomly distributed linker lengths in the range of 18-72 bp. We show that nonuniform distributions with NFRs enhance flexibility and encourage kb-range contacts. NFRs between neighboring gene segments diminish short-range contacts between flanking nucleosomes, while enhancing kb-range contacts via hierarchical looping. We also demonstrate that variances in linker lengths enhance such contacts. In particular, moderate sized variations in fiber linker lengths (∼27 bp) encourage long-range contacts in randomly distributed linker length fibers. Our work underscores the importance of linker length patterns, alongside bound proteins, in biological regulation. Contacts formed by kb-range chromatin folding are crucial to gene activity. Because we find that special linker length distributions in living systems promote kb contacts, our work suggests ways to manipulate these patterns for regulation of gene activity.

  2. Spermiogenesis and chromatin condensation in the common tree shrew, Tupaia glis.

    PubMed

    Suphamungmee, Worawit; Wanichanon, Chaitip; Vanichviriyakit, Rapeepun; Sobhon, Prasert

    2008-03-01

    We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80-100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15-16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12-16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells.

  3. Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

    PubMed Central

    Chen, Chen; Lim, Hong Hwa; Shi, Jian; Tamura, Sachiko; Maeshima, Kazuhiro; Surana, Uttam; Gan, Lu

    2016-01-01

    Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin—nucleosomes—are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an “open” configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome–nucleosome associations. PMID:27605704

  4. An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length.

    PubMed

    Wong, Hua; Victor, Jean-Marc; Mozziconacci, Julien

    2007-09-12

    In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.

  5. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  6. Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template

    PubMed Central

    Hu, Yan; Kireev, Igor; Plutz, Matt; Ashourian, Nazanin

    2009-01-01

    The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization. PMID:19349581

  7. Full-field hard x-ray microscopy below 30 nm : a challenging nanofabrication achievement.

    SciTech Connect

    Chen, Y.; Lo, T.; Chu, Y.; Yi, J.; Liu, C.; Wang, J.; Wang, C.; Chiu, C.; Hua, T.; Hwu, Y.; Shen, Q.; Yin, G.; Liang, K.; Lin, H.; Je, J.; Margaritondo, G.; X-Ray Science Division; Academia Sinica; Tatung Univ.; National Tsing Hua Univ.; National Taiwan Ocean Univ.; National Synchrotron Radiation Research Center; Pohang Univ. of Science and Technology; Ecole Polytechnique Federale de Lausanne

    2008-01-01

    The fabrication of devices to focus hard x-rays is one of the most difficult--and important--challenges in nanotechnology. Here we show that Fresnel zone plates combining 30 nm external zones and a high aspect ratio finally bring hard x-ray microscopy beyond the 30 nm Rayleigh spatial resolution level and measurable spatial frequencies down to 20-23 nm feature size. After presenting the overall nanofabrication process and the characterization test results, we discuss the potential research impact of these resolution levels.

  8. Measurement of local chromatin compaction by spectral precision distance microscopy

    NASA Astrophysics Data System (ADS)

    Rauch, Joachim; Hausmann, Michael; Solovei, Irina; Horsthemke, Bernhard; Cremer, Thomas; Cremer, Christoph G.

    2000-12-01

    Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.

  9. Chromatin Topological Transitions

    NASA Astrophysics Data System (ADS)

    Lavelle, C.; Bancaud, A.; Recouvreux, P.; Barbi, M.; Victor, J.; Viovy, J.

    DNA transaction events occurring during a cell cycle (transcription,repair, replication) are always associated with severe topological constraints on the double helix. However, since nuclear DNA is bound to various proteins (including histones) that control its accessibility and 3D organization, these topological constraints propagate or accumulate on a chromatin substrate. This paper focuses on chromatin fiber response to physiological mechanical constraints expected to occur during transcription elongation. We will show in particular how recent single molecule techniques help us to understand how chromatin conformational dynamics could manage harsh DNA supercoiling changes.

  10. New filter rating method in practice for sub-30-nm lithography process filter

    NASA Astrophysics Data System (ADS)

    Umeda, Toru; Mizuno, Takehito; Tsuzuki, Shuichi; Numaguchi, Toru

    2010-04-01

    A new method for rating retention in lithography process filters has been developed. The method employs a gold nanoparticle contaminant challenge, inductively coupled plasma mass spectrometry as a concentration detector, and dynamic light scattering as a particle size detector, all of which enable accurate, reliable filter retention rating below 30 nm. There is good agreement between results obtained with the new method and results obtained with a conventional polystyrene latex bead challenge. A filter that was rated at 10 nm using extrapolative methods was confirmed to be 10 nm using the new challenge test. Microbridge removal efficiency of polyethylene filters rated by the new method was studied in a 193 nm (dry) lithography process and the new method was verified. When applied to commercially available filters that are rated below 30 nm, the new method revealed significant differences in removal efficiency among similarly labeled filters.

  11. Statistical Mechanics of Nucleosomes Constrained by Higher-Order Chromatin Structure

    NASA Astrophysics Data System (ADS)

    Chereji, Răzvan V.; Morozov, Alexandre V.

    2011-07-01

    Eukaryotic DNA is packaged into chromatin: one-dimensional arrays of nucleosomes separated by stretches of linker DNA are folded into 30-nm chromatin fibers which in turn form higher-order structures (Felsenfeld and Groudine in Nature 421:448, 2003). Each nucleosome, the fundamental unit of chromatin, has 147 base pairs (bp) of DNA wrapped around a histone octamer (Richmond and Davey in Nature 423:145, 2003). In order to describe how chromatin fiber formation affects nucleosome positioning and energetics, we have developed a thermodynamic model of finite-size particles with effective nearest-neighbor interactions and arbitrary DNA-binding energies. We show that both one- and two-body interactions can be extracted from one-particle density profiles based on high-throughput maps of in vitro or in vivo nucleosome positions. Although a simpler approach that neglects two-body interactions (even if they are in fact present in the system) can be used to predict sequence determinants of nucleosome positions, the full theory is required to disentangle one- and two-body effects. Finally, we construct a minimal model in which nucleosomes are positioned primarily by steric exclusion and two-body interactions rather than intrinsic histone-DNA sequence preferences. The model reproduces nucleosome occupancy patterns observed over transcribed regions in living cells.

  12. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Colvin, M E; Thelen, M P; Noy, A

    2004-01-06

    The DNA in eukaryotic cells is tightly packaged as chromatin through interactions with histone proteins to form nucleosomes. These nucleosomes are themselves packed together through interactions with linker histone and non-histone proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the chromatin fiber must be remodeled such that the necessary enzymes can access the DNA. The structure of the chromatin fiber beyond the level of the single nucleosome and the structural changes which accompany the remodeling process are poorly understood. We are studying the structures and forces behind the remodeling process through the use of atomic force microscopy (AFM). This allows both high-resolution imaging of the chromatin, and manipulation of individual fibers. Pulling a single chromatin fiber apart using the AFM tip yields information on the forces which hold the structure together. We have isolated chromatin fibers from chicken erythrocytes and Chinese hamster ovary cell lines. AFM images of these fibers will be presented, along with preliminary data from the manipulation of these fibers using the AFM tip. The implications of these data for the structure of chromatin undergoing the remodeling process are discussed.

  13. In Vitro Effect of 30 nm Silver Nanoparticles on Adipogenic Differentiation of Human Mesenchymal Stem Cells.

    PubMed

    He, Wei; Kienzle, Arne; Liu, Xujie; Müller, Werner E G; Elkhooly, Tarek A; Feng, Qingling

    2016-03-01

    With the combined use of silver nanoparticles (Ag NPs) and human bone marrow derived mesenchymal stem cells (hMSCs) in bone tissue engineering, more knowledge of the effects of Ag NPs on hMSCs is required. Up to date, researches mainly focused on the cytotoxicity and genotoxicity of Ag NPs, only few studies discussed their influence on the differentiation of stem cells, especially adipogenic differentiation. In the present study, we investigated the in vitro uptake of 30 nm PVP-coated Ag NPs in hMSCs and their effects on cell viability, cell morphology and adipogenic differentiation of hMSCs. HMSCs were exposed to Ag NPs at concentrations of 25 and 50 μg/mL for 24 hours and at concentrations of 5 and 10 μg/mL throughout the whole differentiation period. Results of cell viability showed that Ag NPs caused time- and dose-dependent toxicity in hMSCs. Transmission electron microscopy (TEM) confirmed the uptake of Ag NPs into cytoplasm of hMSCs. No influence on cell morphology was observed. The 30 nm sized Ag NPs had no effects on adiponectin secretion, lipid droplet formation and the expression of adipogenic marker genes. It is concluded that under our experimental conditions, 30 nm PVP-coated Ag NPs do not influence the adipogenic differentiation of hMSCs in vitro. The present results provide a reference for the usage of 30 nm Ag NPs in the presence of hMSCs in bone tissue engineering.

  14. UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro

    SciTech Connect

    Gale, J.M.; Smerdon, M.J.

    1988-09-20

    We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.

  15. Fabrication of 30 nm inter-electrode gap co-planar tunnel junctions with buried electrodes

    NASA Astrophysics Data System (ADS)

    Itoua, S.; Joachim, C.; Rousset, B.; Fabre, N.

    1994-05-01

    Co-planar tunnel junctions with a gap length in the 30 nm range have been fabricated using a 20 keV scanning electron microscope and a Au-Pd lift-off. The junction electrodes are less than 200 nm in width and are buried in the SiO2 substrate. This makes the gap surface accessible for atomic force microscope characterization and for local modification. Des jonctions tunnels co-planaires avec une largeur de coupure inférieure à 30 nm ont été fabriquées en utilisant un masqueur électronique à 20 keV et un procédé de lift-off d'un alliage Au-Pd. Les électrodes de la jonction ont moins de 200 nm de largeur et sont enterrées à la surface de SiO2. La mesure de la topographie de la surface de la coupure avec un microscope à force atomique montre une rugosité de moins de 1 nm.

  16. TOPICAL REVIEW: The physics of chromatin

    NASA Astrophysics Data System (ADS)

    Schiessel, Helmut

    2003-05-01

    Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

  17. Mesoscale Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    Schlick, Tamar

    2009-03-01

    Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

  18. Characterization of promising resist platforms for sub-30-nm HP manufacturability and EUV CAR extendibility study

    NASA Astrophysics Data System (ADS)

    Koh, Chawon; Georger, Jacque; Ren, Liping; Huang, George; Goodwin, Frank; Wurm, Stefan; Ashworth, Dominic; Montgomery, Warren; Pierson, Bill; Park, Joo-on; Naulleau, Patrick

    2010-04-01

    Extreme ultraviolet lithography (EUVL) is the most effective way to print sub-30 nm features. We characterized EUVL readiness of the three major resist platforms for sub-30 nm half-pitch (HP) manufacturability using a full-field ASML alpha demo tool (ADT) scanner and studied the extendibility of EUV chemically amplified resist (CAR). Based on an "M-factor" analysis, which shows the maturity of EUV resist for 28 nm HP manufacturability, a polymer-bound photoacid generator (PAG) resist was 78% ready, a PHS hybrid resist was 81%, and a molecular glass EUV resist was 58%. The polymer-bound resist showed good resolution for 25 nm HP using the ASML ADT. It also demonstrated fair linewidth roughness (LWR) and a good lithographic process margin of 18% exposure latitude (EL) and 160 nm depth of field (DOF) for 28 nm HP patterning compared with the other resist platforms, but its resist collapse and etch resistance need to be improved for manufacturability. PHS hybrid resist showed a fair etch resistance and resist collapse performance compared to the other resist platforms, but LWR needs to be improved. The molecular resist needs to mature further, especially in resist collapse and iso-dense (ID) bias. When considering its many strong points and control of lower acid diffusion, the polymer-bound PAG resist appears to be the most suitable platform for manufacturability and EUV CAR extension. We therefore would like to encourage the development of next generation polymer-bound PAG resist with a higher etch resistance. A process window of 80 nm DOF was demonstrated for 26 nm HP patterning and a measurable DOF for 25 nm HP was achieved with the polymer-bound PAG resist. Resist collapse and LWR are major issues for 22 nm HP patterning in manufacturing. LWR improvements were achieved with various techniques, and resist collapse was greatly improved with a novel approach that uses a residual resist layer. 16 nm HP line/space (L/S) image modulation and 18 nm HP resolution were

  19. Neutron scattering studies on chromatin higher-order structure

    SciTech Connect

    Graziano, V.; Gerchman, S.E.; Schneider, D.K.; Ramakrishnan, V.

    1994-12-31

    We have been engaged in studies of the structure and condensation of chromatin into the 30nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6-7 nucleosomes/11 nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5-7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

  20. Octaarginine Labelled 30 nm Gold Nanoparticles as Agents for Enhanced Radiotherapy

    NASA Astrophysics Data System (ADS)

    Latimer, Caitlin Louise

    Traditional radiation therapy is limited by the radiotoxic effects on surrounding healthy tissues. This project investigated the use of a gold nanoparticle (AuNP) conjugated to a cell-penetrating peptide (CPP) to increase tumour cell death during radiotherapy by maximizing the cellular import of the gold nanoparticles. ˜8300 octaarginine CPPs were coupled per 30 nm AuNP through poly(ethylene glycol) spacers (AuNP-PEG-CPP). The CPPs enhanced the internalization of the AuNPs into three human breast cancer cell lines by a factor >2 as compared to untargeted AuNPs. Cells were treated with AuNP-PEG-CPP for 24 hours, prior to radiotherapy and their long-term proliferation was assessed in clonogenic assays. The increased internalization of AuNPs by the CPPs resulted in greater cell death following exposure to 300 kVp radiotherapy, by a dose enhancement factors between 1.3 and 2.1 depending on the cell line. These findings illustrate the potential of using AuNP-CPPs to enhance radiotherapy in patients.

  1. Flexible and dynamic nucleosome fiber in living mammalian cells.

    PubMed

    Nozaki, Tadasu; Kaizu, Kazunari; Pack, Chan-Gi; Tamura, Sachiko; Tani, Tomomi; Hihara, Saera; Nagai, Takeharu; Takahashi, Koichi; Maeshima, Kazuhiro

    2013-01-01

    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/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.

  2. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    SciTech Connect

    Oeberg, Christine; Belikov, Sergey

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, {Delta}N-hH1.4, were compared. Black-Right-Pointing-Pointer Both histones bind to chromatin, however, {Delta}N-hH1.4 displays lower binding affinity. Black-Right-Pointing-Pointer Interaction of {Delta}N-hH1.4 with chromatin includes a significant unspecific component. Black-Right-Pointing-Pointer N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain ({Delta}N-hH1.4). The {Delta}N-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that {Delta}N-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  3. Advanced self-aligned double patterning development for sub-30-nm DRAM manufacturing

    NASA Astrophysics Data System (ADS)

    Shiu, Weicheng; Liu, Hung Jen; Wu, Jan Shiun; Tseng, Tsu-Li; Liao, Chun Te; Liao, Chien Mao; Liu, Jerry; Wang, Troy

    2009-03-01

    /or fancy integration approaches. In our study we purposely apply a more "classical" and relatively conservative integration scheme, with all unit process steps long proven in previous volume production. By carefully optimizing the relative CMP, films deposition, etch and cleaning processes, we are able to demonstrate 30nm line/space patterns by an NA 0.93 dry 193nm scanner with optimal CDU better than 3nm and high frequency line edge roughness (LER) close to 2nm/side. Additionally, by analyzing wafer quality for alignment and alignment residual in various alignment & overlay mark designs, projected residual overlay as little as 4nm can be readily obtained.

  4. Chromatin Dynamics

    PubMed Central

    Hübner, Michael R.; Spector, David L.

    2010-01-01

    The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes. PMID:20462379

  5. Computational strategies to address chromatin structure problems

    NASA Astrophysics Data System (ADS)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

    While the genetic information is contained in double helical DNA, gene expression is a complex multilevel process that involves various functional units, from nucleosomes to fully formed chromatin fibers accompanied by a host of various chromatin binding enzymes. The chromatin fiber is a polymer composed of histone protein complexes upon which DNA wraps, like yarn upon many spools. The nature of chromatin structure has been an open question since the beginning of modern molecular biology. Many experiments have shown that the chromatin fiber is a highly dynamic entity with pronounced structural diversity that includes properties of idealized zig-zag and solenoid models, as well as other motifs. This diversity can produce a high packing ratio and thus inhibit access to a majority of the wound DNA. Despite much research, chromatin’s dynamic structure has not yet been fully described. Long stretches of chromatin fibers exhibit puzzling dynamic behavior that requires interpretation in the light of gene expression patterns in various tissue and organisms. The properties of chromatin fiber can be investigated with experimental techniques, like in vitro biochemistry, in vivo imagining, and high-throughput chromosome capture technology. Those techniques provide useful insights into the fiber’s structure and dynamics, but they are limited in resolution and scope, especially regarding compact fibers and chromosomes in the cellular milieu. Complementary but specialized modeling techniques are needed to handle large floppy polymers such as the chromatin fiber. In this review, we discuss current approaches in the chromatin structure field with an emphasis on modeling, such as molecular dynamics and coarse-grained computational approaches. Combinations of these computational techniques complement experiments and address many relevant biological problems, as we will illustrate with special focus on epigenetic modulation of chromatin structure.

  6. Chromatin remodeling: nucleosomes bulging at the seams.

    PubMed

    Peterson, Craig L

    2002-04-02

    ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.

  7. Nucleosomes, Linker DNA, and Linker Histone form a Unique Structural Motif that Directs the Higher-Order Folding and Compaction of Chromatin

    NASA Astrophysics Data System (ADS)

    Bednar, Jan; Horowitz, Rachel A.; Grigoryev, Sergei A.; Carruthers, Lenny M.; Hansen, Jeffrey C.; Koster, Abraham J.; Woodcock, Christopher L.

    1998-11-01

    The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈ 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈ 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

  8. Thickness dependence of planar Hall resistance and field sensitivity in NiO(30 nm)/NiFe( t) bilayers

    NASA Astrophysics Data System (ADS)

    Kim, D. Y.; Kim, C. G.; Park, B. S.; Park, C. M.

    2000-06-01

    We measured the planar Hall resistance (PHR) profiles in NiO (30 nm)/NiFe( t) bilayers for t=5, 10, 20 and 30 nm and analyzed its field sensitivity in terms of exchange-coupling field and anisotropy constant. The measured PHR shows linear field dependence at near H=0 as well as small hysteresis. The linear field range Δ H and resistance change, Δ R= R∥- R⊥, decrease with the NiFe thickness, where Δ H is calculated to be proportional to the anisotropy constant Ku and exchange-coupling field Hex. However, the field sensitivity Δ R/Δ H shows a maximum value at t=20 nm; where Ku is the minimum. The PHR has the advantage of a linear response at the operating field range and can be used for a recording read-out head and related applications.

  9. Teaching resources. Chromatin remodeling.

    PubMed

    Lue, Neal F

    2005-07-26

    This Teaching Resource provides lecture notes and slides for a class covering chromatin remodeling mechanisms and is part of the course "Cell Signaling Systems: a Course for Graduate Students." The lecture begins with a discussion of chromatin organization and then proceeds to describe the process of chromatin remodeling through a review of chromatin remodeling complexes and methods used to study their function.

  10. Chromatin as an expansive canvas for chemical biology.

    PubMed

    Fierz, Beat; Muir, Tom W

    2012-04-17

    Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.

  11. Orientation of nucleosomes and linker DNA in calf thymus chromatin determined by photochemical dichroism

    NASA Astrophysics Data System (ADS)

    Mitra, Sekhar; Sen, Dipankar; Crothers, Donald M.

    1984-03-01

    The dichroism for photochemical attachment of a psoralen derivative to Mg2+ -stabilized chromatin fibres is used to deduce the orientation of nucleosomal disks and linker DNA in the 30-nm fibre. The new technique of photochemical electric dichroism should have general applicability to problems of nucleic acid organization in cellular subunits and viruses.

  12. Cryogenic ion implantation near amorphization threshold dose for halo/extension junction improvement in sub-30 nm device technologies

    SciTech Connect

    Park, Hugh; Todorov, Stan; Colombeau, Benjamin; Rodier, Dennis; Kouzminov, Dimitry; Zou Wei; Guo Baonian; Khasgiwale, Niranjan; Decker-Lucke, Kurt

    2012-11-06

    We report on junction advantages of cryogenic ion implantation with medium current implanters. We propose a methodical approach on maximizing cryogenic effects on junction characteristics near the amorphization threshold doses that are typically used for halo implants for sub-30 nm technologies. BF{sub 2}{sup +} implant at a dose of 8 Multiplication-Sign 10{sup 13}cm{sup -2} does not amorphize silicon at room temperature. When implanted at -100 Degree-Sign C, it forms a 30 - 35 nm thick amorphous layer. The cryogenic BF{sub 2}{sup +} implant significantly reduces the depth of the boron distribution, both as-implanted and after anneals, which improves short channel rolloff characteristics. It also creates a shallower n{sup +}-p junction by steepening profiles of arsenic that is subsequently implanted in the surface region. We demonstrate effects of implant sequences, germanium preamorphization, indium and carbon co-implants for extension/halo process integration. When applied to sequences such as Ge+As+C+In+BF{sub 2}{sup +}, the cryogenic implants at -100 Degree-Sign C enable removal of Ge preamorphization, and form more active n{sup +}-p junctions and steeper B and In halo profiles than sequences at room temperature.

  13. Cryogenic ion implantation near amorphization threshold dose for halo/extension junction improvement in sub-30 nm device technologies

    NASA Astrophysics Data System (ADS)

    Park, Hugh; Todorov, Stan; Colombeau, Benjamin; Rodier, Dennis; Kouzminov, Dimitry; Zou, Wei; Guo, Baonian; Khasgiwale, Niranjan; Decker-Lucke, Kurt

    2012-11-01

    We report on junction advantages of cryogenic ion implantation with medium current implanters. We propose a methodical approach on maximizing cryogenic effects on junction characteristics near the amorphization threshold doses that are typically used for halo implants for sub-30 nm technologies. BF2+ implant at a dose of 8×1013cm-2 does not amorphize silicon at room temperature. When implanted at -100°C, it forms a 30 - 35 nm thick amorphous layer. The cryogenic BF2+ implant significantly reduces the depth of the boron distribution, both as-implanted and after anneals, which improves short channel rolloff characteristics. It also creates a shallower n+-p junction by steepening profiles of arsenic that is subsequently implanted in the surface region. We demonstrate effects of implant sequences, germanium preamorphization, indium and carbon co-implants for extension/halo process integration. When applied to sequences such as Ge+As+C+In+BF2+, the cryogenic implants at -100°C enable removal of Ge preamorphization, and form more active n+-p junctions and steeper B and In halo profiles than sequences at room temperature.

  14. Synthesis of highly ordered 30 nm NiFe2O4 particles by the microwave-combustion method

    NASA Astrophysics Data System (ADS)

    Mahmoud, M. H.; Elshahawy, A. M.; Makhlouf, Salah A.; Hamdeh, H. H.

    2014-11-01

    NiFe2O4 of 30 nm average size was synthesized by microwave combustion and subsequent solid state reaction at 1273 K. The materials were characterized by X-ray diffraction, TEM, vibrating sample magnetometery and Mössbauer spectroscopy. The microwave combustion produced materials were comprised chemically of ferrites and a smaller amount of hematite. The NiFe2O4 particles have the cubic spinel structure with crystallites of sizes less than 10 nm, and were found to have low magnetization, and essentially no hysteresis loop; characteristics of superparamagnetism. Upon annealing at temperatures 973 K and below, crystallite growth was accompanied by increase in both coercive field and magnetization. The coercive field was a maximum for the sample annealed at 973 K. On the other hand, crystallite growth at higher annealing temperatures yielded mainly ferrites and improvement in soft magnetic properties. Mössbauer and magnetization measurements indicate that the fine NiFe2O4 particles produced at the annealing temperature of 1273 K are in good chemical and magnetic order, excluding the spins arrangement at the surface of the particles which show spin glass-like behavior.

  15. The chromatin regulatory code: Beyond a histone code

    NASA Astrophysics Data System (ADS)

    Lesne, A.

    2006-03-01

    In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of “chromatin code” introduced and elaborated in this paper is to be preferred to that of “histone code”. Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.

  16. Chromatin enrichment for proteomics

    PubMed Central

    Kustatscher, Georg; Wills, Karen L. H.; Furlan, Cristina; Rappsilber, Juri

    2015-01-01

    During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a ‘snapshot’ of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses. PMID:25101823

  17. Physical properties of unacetylated chromatin as examined by magnetic tweezers

    NASA Astrophysics Data System (ADS)

    McGill, Kerry; Dunlap, David; Lucchesi, John

    2011-10-01

    As the source of genetic material, DNA is involved in a variety of biological processes like transcription, cell replication, and more. In these processes, DNA is manipulated into different structures and is subjected to different levels of physical force on a molecular scale. When tension is applied to one hierarchical structure called chromatin, it appears to behave like a Hookian spring. The base component of chromatin is a nucleosome, which is constructed when DNA coils around octamers of histone proteins. The histones can become acetylated---a chemical process in which an acetyl functional group attaches to amino acids of the histones, often lysines. Acetylation may loosen chromatin's coils and therefore lower the amount of tension required to stretch the chromatin. Comparing the levels of tension required to stretch acetylated chromatin could reveal, directly, physical differences in the chromatin fiber that bear ion the function of the DNA molecule. Work presented will be the investigation of unacetylated chromatin.

  18. Distribution, elimination, and biopersistence to 90 days of a systemically introduced 30 nm ceria-engineered nanomaterial in rats.

    PubMed

    Yokel, Robert A; Au, Tu C; MacPhail, Robert; Hardas, Sarita S; Butterfield, D Allan; Sultana, Rukhsana; Goodman, Michael; Tseng, Michael T; Dan, Mo; Haghnazar, Hamed; Unrine, Jason M; Graham, Uschi M; Wu, Peng; Grulke, Eric A

    2012-05-01

    Nanoceria is used as a catalyst in diesel fuel, as an abrasive in printed circuit manufacture, and is being pursued as an antioxidant therapeutic. Our objective is to extend previous findings showing that there were no reductions of cerium in organs of the mononuclear phagocyte (reticuloendothelial) system up to 30 days after a single nanoscale ceria administration. An ~5% aqueous dispersion of citrate-stabilized 30 nm ceria, synthesized and characterized in-house, or vehicle, was iv infused into rats terminated 1, 7, 30, or 90 days later. Cageside observations were obtained daily, body weight weekly. Daily urinary and fecal cerium outputs were quantified for 2 weeks. Nine organs were weighed and samples collected from 14 tissues/organs/systems, blood and cerebrospinal fluid for cerium determination. Histology and oxidative stress were assessed. Less than 1% of the nanoceria was excreted in the first 2 weeks, 98% in feces. Body weight gain was initially impaired. Spleen weight was significantly increased in some ceria-treated groups, associated with abnormalities. Ceria was primarily retained in the spleen, liver, and bone marrow. There was little decrease of ceria in any tissue over the 90 days. Granulomas were observed in the liver. Time-dependent oxidative stress changes were seen in the liver and spleen. Nanoscale ceria was persistently retained by organs of the mononuclear phagocyte system, associated with adverse changes. The results support concern about the long-term fate and adverse effects of inert nanoscale metal oxides that distribute throughout the body, are persistently retained, and produce adverse changes.

  19. Fiber

    MedlinePlus

    ... it can help with weight control. Fiber aids digestion and helps prevent constipation . It is sometimes used ... fiber attracts water and turns to gel during digestion. This slows digestion. Soluble fiber is found in ...

  20. Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes*

    PubMed Central

    Hoffman, Elizabeth A.; Frey, Brian L.; Smith, Lloyd M.; Auble, David T.

    2015-01-01

    Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function. PMID:26354429

  1. Long range chromatin organization

    PubMed Central

    Acuña, Luciana I Gómez; Kornblihtt, Alberto R

    2014-01-01

    Splicing is a predominantly co-transcriptional process that has been shown to be tightly coupled to transcription. Chromatin structure is a key factor that mediates this functional coupling. In light of recent evidence that shows the importance of higher order chromatin organization in the coordination and regulation of gene expression, we discuss here the possible roles of long-range chromatin organization in splicing and alternative splicing regulation. PMID:25764333

  2. Nucleosome positioning and composition modulate in silico chromatin flexibility.

    PubMed

    Clauvelin, N; Lo, P; Kulaeva, O I; Nizovtseva, E V; Diaz-Montes, J; Zola, J; Parashar, M; Studitsky, V M; Olson, W K

    2015-02-18

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and

  3. Nucleosome positioning and composition modulate in silico chromatin flexibility

    NASA Astrophysics Data System (ADS)

    Clauvelin, N.; Lo, P.; Kulaeva, O. I.; Nizovtseva, E. V.; Diaz-Montes, J.; Zola, J.; Parashar, M.; Studitsky, V. M.; Olson, W. K.

    2015-02-01

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes—the familiar assemblies of ˜150 DNA base pairs and eight histone proteins—found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the ‘local’ inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome

  4. Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture.

    PubMed

    Yu, Miao; Huang, Shaohui; Yu, Kevin Jun; Clyne, Alisa Morss

    2012-01-01

    Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

  5. Chromatin Ring Formation at Plant Centromeres

    PubMed Central

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

    We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants. PMID:26913037

  6. Chromatin associations in Arabidopsis interphase nuclei.

    PubMed

    Schubert, Veit; Rudnik, Radoslaw; Schubert, Ingo

    2014-01-01

    The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analyzed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fiber movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns. Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its 10 centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei.

  7. The dynamics of HMG protein–chromatin interactions in living cells1

    PubMed Central

    Gerlitz, Gabi; Hock, Robert; Ueda, Tetsuya; Bustin, Michael

    2012-01-01

    The dynamic interaction between nuclear proteins and chromatin leads to the functional plasticity necessary to mount adequate responses to regulatory signals. Here, we review the factors regulating the chromatin interactions of the high mobility group proteins (HMGs), an abundant and ubiquitous superfamily of chromatin-binding proteins in living cells. HMGs are highly mobile and interact with the chromatin fiber in a highly dynamic fashion, as part of a protein network. The major factors that affect the binding of HMGs to chromatin are operative at the level of the single nucleosome. These factors include structural features of the HMGs, competition with other chromatin-binding proteins for nucleosome binding sites, complex formation with protein partners, and post-translational modifications in the protein or in the chromatin-binding sites. The versatile modulation of the interaction between HMG proteins and chromatin plays a role in processes that establish the cellular phenotype. PMID:19234529

  8. Dynamic structures of intact chicken erythrocyte chromatins as studied by 1H-31P cross-polarization NMR.

    PubMed Central

    Akutsu, H; Nishimoto, S; Kyogoku, Y

    1994-01-01

    The dynamic properties of DNA in intact chicken erythrocyte cells, nuclei, nondigested chromatins, digested soluble chromatins, H1, H5-depleted soluble chromatins and nucleosome cores were investigated by means of single-pulse and 1H-31P cross-polarization NMR. The temperature dependence of the phosphorus chemical shift anisotropy was identical for the former three in the presence of 3 mM MgCl2, suggesting that the local higher order structure is identical for these chromatins. The intrinsic phosphorus chemical shift anisotropy of the nucleosome cores was -159 ppm. The chemical shift anisotropy of DNA in the chromatins can be further averaged by the motion of the linker DNA. The spin-lattice relaxation time in the rotating frame of the proton spins (T1p) of the nondigested chromatins was measured at various locking fields. The result was analyzed on the assumption of the isotropic motion to get a rough value of the correlation time of the motion efficient for the relaxation, which was eventually ascribed to the segmental motion of the linker DNA with restricted amplitude. The 30 nm filament structure induced by NaCl was shown to be dynamically different from that induced by MgCl2. Side-by-side compaction of 30-nm filaments was suggested to be induced in the MgCl2 concentration range higher than 0.3 mM. Biological significance of the dynamic structure was discussed in connection with the results obtained. PMID:7948693

  9. Human sperm chromatin stabilization: a proposed model including zinc bridges.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2010-01-01

    The primary focus of this review is to challenge the current concepts on sperm chromatin stability. The observations (i) that zinc depletion at ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents and (ii) that the human sperm chromatin contains one zinc for every protamine for every turn of the DNA helix suggest an alternative model for sperm chromatin structure may be plausible. An alternative model is therefore proposed, that the human spermatozoon could at ejaculation have a rapidly reversible zinc dependent chromatin stability: Zn(2+) stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism, the formation of zinc bridges with protamine thiols of cysteine and potentially imidazole groups of histidine. Extraction of zinc enables two biologically totally different outcomes: immediate decondensation if chromatin fibers are concomitantly induced to repel (e.g. by phosphorylation in the ooplasm); otherwise freed thiols become committed into disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be accomplished by the seminal vesicular fluid. Collection of the ejaculate in one single container causes abnormal contact between spermatozoa and seminal vesicular fluid affecting the sperm chromatin stability. There are men in infertile couples with low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

  10. Dietary control of chromatin

    PubMed Central

    Huang, Zhiguang; Cai, Ling; Tu, Benjamin P

    2015-01-01

    Organisms must be able to rapidly alter gene expression in response to changes in their nutrient environment. This review summarizes evidence that epigenetic modifications of chromatin depend on particular metabolites of intermediary metabolism, enabling the facile regulation of gene expression in tune with metabolic state. Nutritional or dietary control of chromatin is an often-overlooked, yet fundamental regulatory mechanism directly linked to human physiology. Nutrient-sensitive epigenetic marks are dynamic, suggesting rapid turnover, and may have functions beyond the regulation of gene transcription, including pH regulation and as carbon sources in cancer cells. PMID:26094239

  11. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  12. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  13. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

  14. Mechanobiology of Chromatin and the Nuclear Interior.

    PubMed

    Spagnol, Stephen T; Armiger, Travis J; Dahl, Kris Noel

    2016-06-01

    The view of the cell nucleus has evolved from an isolated, static organelle to a dynamic structure integrated with other mechanical elements of the cell. Both dynamics and integration appear to contribute to a mechanical regulation of genome expression. Here, we review physical structures inside the nucleus at different length scales and the dynamic reorganization modulated by cellular forces. First, we discuss nuclear organization focusing on self-assembly and disassembly of DNA structures and various nuclear bodies. We then discuss the importance of connections from the chromatin fiber through the nuclear envelope to the rest of the cell as they relate to mechanobiology. Finally, we discuss how cell stimulation, both chemical and physical, can alter nuclear structures and ultimately cellular function in healthy cells and in some model diseases. The view of chromatin and nuclear bodies as mechanical entities integrated with force generation from the cytoskeleton combines polymer physics with cell biology and medicine.

  15. Interaction of chromatin with NaCl and MgCl2. Solubility and binding studies, transition to and characterization of the higher-order structure.

    PubMed

    Ausio, J; Borochov, N; Seger, D; Eisenberg, H

    1984-08-15

    Chicken erythrocyte chromatin containing histones H1 and H5 was carefully separated into a number of well-characterized fractions. A distinction could be made between chromatin insoluble in NaCl above about 80 mM, and chromatin soluble at all NaCl concentrations. Both chromatin forms were indistinguishable electrophoretically and both underwent the transition from the low salt "10 nm" coil to the "30 nm" higher-order structure solenoid by either raising the MgCl2 concentration to about 0.3 mM or the NaCl concentration to about 75 mM. The transitions were examined in detail by elastic light-scattering procedures. It could be shown that the 10 nm form is a flexible coil. For the 30 nm solenoid, the assumption of a rigid cylindrical structure was in good agreement with 5.7 nucleosomes per helical turn. However, disagreement of calculated frictional parameters with values derived from quasielastic light-scattering and sedimentation introduced the possibility that the higher-order structure, under these conditions, is more extended, flexible, or perhaps a mixture of structures. Values for density and refractive index increments of chromatin are also given. To understand the interaction of chromatin with NaCl and with MgCl2, a number of experiments were undertaken to study solubility, precipitation, conformational transitions and binding of ions over a wide range of experimental conditions, including chromatin concentration.

  16. Cohesin organizes chromatin loops at DNA replication factories

    PubMed Central

    Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

    2010-01-01

    Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

  17. Chromatin structure in bands and interbands of polytene chromosomes imaged by atomic force microscopy.

    PubMed

    de Grauw, C J; Avogadro, A; van den Heuvel, D J; vd Werf, K O; Otto, C; Kraan, Y; van Hulst, N F; Greve, J

    1998-01-01

    Polytene chromosomes from Drosophila melanogaster, observed from squash preparations, and chromosomes from Chironomus thummi thummi, investigated under physiological conditions, are imaged using an Atomic Force Microscope. Various chromatin fiber structures can be observed with high detail in fixed chromosomes and correspond to structures which are also observed in chromosomes of diploid cells. Unfixed chromosomes can be imaged in buffer and show less fiber-like details because of the inherent soft nature of the chromatin material.

  18. Fiber Optic Microphone

    NASA Technical Reports Server (NTRS)

    Cho, Y. C.; George, Thomas; Norvig, Peter (Technical Monitor)

    1999-01-01

    Research into advanced pressure sensors using fiber-optic technology is aimed at developing compact size microphones. Fiber optic sensors are inherently immune to electromagnetic noise, and are very sensitive, light weight, and highly flexible. In FY 98, NASA researchers successfully designed and assembled a prototype fiber-optic microphone. The sensing technique employed was fiber optic Fabry-Perot interferometry. The sensing head is composed of an optical fiber terminated in a miniature ferrule with a thin, silicon-microfabricated diaphragm mounted on it. The optical fiber is a single mode fiber with a core diameter of 8 micron, with the cleaved end positioned 50 micron from the diaphragm surface. The diaphragm is made up of a 0.2 micron thick silicon nitride membrane whose inner surface is metallized with layers of 30 nm titanium, 30 nm platinum, and 0.2 micron gold for efficient reflection. The active sensing area is approximately 1.5 mm in diameter. The measured differential pressure tolerance of this diaphragm is more than 1 bar, yielding a dynamic range of more than 100 dB.

  19. Demonstration of saturated tabletop soft x-ray lasers at 5 Hz repetition rate in transitions of Ne-like ions with wavelengths near 30 nm

    NASA Astrophysics Data System (ADS)

    Wang, Yong; Shlyaptsev, Vyacheslav N.; Rocca, Jorge J.

    2005-10-01

    Recent experiments have demonstrated that the laser pump energy required to operate collisional soft x-ray lasers in the gain saturated regime can be significantly reduced by directing the heating pulse into the plasma at grazing incidence for a more efficient energy deposition [1-2]. Optimization of the incidence angle led to gain-saturated operation at 5Hz repetition rate in several transitions of Ni-like ions at wavelengths ranging from 18.9nm to 13.2nm [3]. We report saturated high repetition rate laser-pumped table-top soft x-ray lasers in Ne-like ions at wavelengths near 30nm. Gain-saturated lasers operating at 5Hz repetition rate were obtained in Ne-like Ti at 32.6nm and in Ne-like V at 30.4nm heating plasmas with laser pulses of ˜1J and 8ps impinging at 20^o grazing incidence. Average powers > 1μW were measured. Strong lasing was also observed in Ne-like Cr at 28.6nm. 1. R. Keenan et al, Phys. Rev. Lett., 94, 103901, (2005). 2. B. M. Luther et al, Opt. Lett., 30, 165, (2005). 3. Y. Wang et al, submitted to Phys. Rev. A, (2005).

  20. High-Throughput Synthesis of Lignin Particles (∼30 nm to ∼2 μm) via Aerosol Flow Reactor: Size Fractionation and Utilization in Pickering Emulsions.

    PubMed

    Ago, Mariko; Huan, Siqi; Borghei, Maryam; Raula, Janne; Kauppinen, Esko I; Rojas, Orlando J

    2016-09-07

    An aerosol flow reactor was used for the first time for high-throughput, high yield synthesis of spherical lignin particles with given inherent hydrophilicity, depending on the precursor biomolecule. In situ fractionation via Berner type impactor afforded populations with characteristic sizes ranging from ∼30 nm to 2 μm. The as-produced, dry lignin particles displayed excellent mechanical integrity, even after redispersion under high shear in either mineral oil or water. They were effective in the stabilization of oil-in-water (O/W) Pickering emulsions with tunable droplet size, depending on the dimension of the lignin particles used for emulsification. The emulsion stability correlated with particle concentration as well as the respective lignin type. For the O/W emulsions stabilized with the more hydrophilic lignin particles, negligible changes in phase separation via Ostwald ripening and coalescence were observed over a period of time of more than two months. Together with the fact that the lignin particle concentrations used in emulsification were as low as 0.1%, our results reveal a remarkable ability to endow emulsified systems with high colloidal stability. Overall, we offer a new, high-yield, scalable nanomanufacturing approach to producing dry spherical lignin particles with size control and high production capacity. A number of emerging applications for these organic particles can be envisioned and, as a proof-of-concept, we illustrate here surfactant-free emulsification.

  1. Cas9 Functionally Opens Chromatin

    PubMed Central

    Barkal, Amira A.; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K.; Sherwood, Richard I.

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  2. Molecular Toxicology of Chromatin

    DTIC Science & Technology

    1992-01-01

    FINAL 01 Jan 89 TO 31 Dec 91 4. ITL ANO SUS Y, L RE %UMAS MOLECULAR TOXICOLOGY OF CHROMATIN AFOSR-89-0231 PE - 61102F AUT PR - 2312 TA - A5 Dr Ernest Kun...Waterbury, CT), 2-mercaptoethanol, NAD+, NADPH, nucleo- tides, sodium tungstate , hydrogen peroxide, Tris and MES buffers from Sigma (St. Louis, MO...ml) with sodium tungstate (5.93 g, in 20 ml H20) for 1.5 h followed by extraction of the green product into ethyl acetate, washing with 0.1 N HCl, and

  3. Mapping chromatin modifications in nanochannels

    NASA Astrophysics Data System (ADS)

    Lim, Shuang Fang; Karpusenko, Alena; Riehn, Robert

    2013-03-01

    DNA and chromatin are elongated to a fixed fraction of their contour length when introduced into quasi-1d nanochannels. Because single molecules are analyzed, their hold great potential for the analysis for the genetic analysis of material from single cells. In this study, we have reconstituted chromatin with histones from a variety of sources, and mapped the modification profile of the chromatin. We monitored methylation and acetylation patterns of the histone tail protein residues using fluorescently labelled antibodies. Using those, we distinguished chromatin reconstituted from chicken erythrocytes, calf thymus, and HeLa cells. We discuss prospects for profiling histone modifications for whole chromosomes from single cells.

  4. Chromatin structure in barley nuclei.

    PubMed

    Mithieux, G; Roux, B

    1983-10-03

    In order to study the chromatin structure of a higher plant we used a high-yield method, which allows one to obtain up to 10(9) nuclei/kg fresh barley leaves. Significant amounts of low-ionic-strength-soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro-optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.

  5. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  6. Chromatin condensation during terminal erythropoiesis.

    PubMed

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

  7. CCSI: a database providing chromatin-chromatin spatial interaction information.

    PubMed

    Xie, Xiaowei; Ma, Wenbin; Songyang, Zhou; Luo, Zhenhua; Huang, Junfeng; Dai, Zhiming; Xiong, Yuanyan

    2016-01-01

    Distal regulatory elements have been shown to regulate gene transcription through spatial interactions, and single nucleotide polymorphisms (SNPs) are linked with distal gene expression by spatial proximity, which helps to explain the causal role of disease-associated SNPs in non-coding region. Therefore, studies on spatial interactions between chromatin have created a new avenue for elucidating the mechanism of transcriptional regulation in disease pathogenesis. Recently, a growing number of chromatin interactions have been revealed by means of 3C, 4C, 5C, ChIA-PET and Hi-C technologies. To interpret and utilize these interactions, we constructed chromatin-chromatin spatial interaction (CCSI) database by integrating and annotating 91 sets of chromatin interaction data derived from published literature, UCSC database and NCBI GEO database, resulting in a total of 3,017,962 pairwise interactions (false discovery rate < 0.05), covering human, mouse and yeast. A web interface has been designed to provide access to the chromatin interactions. The main features of CCSI are (i) showing chromatin interactions and corresponding genes, enhancers and SNPs within the regions in the search page; (ii) offering complete interaction datasets, enhancer and SNP information in the download page; and (iii) providing analysis pipeline for the annotation of interaction data. In conclusion, CCSI will facilitate exploring transcriptional regulatory mechanism in disease pathogenesis associated with spatial interactions among genes, regulatory regions and SNPs. Database URL: http://songyanglab.sysu.edu.cn/ccsi.

  8. Structure of chromatin in spermatozoa.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

  9. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  10. Unusual chromatin in human telomeres.

    PubMed Central

    Tommerup, H; Dousmanis, A; de Lange, T

    1994-01-01

    We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere. Images PMID:8065312

  11. Regulation of cellular chromatin state

    PubMed Central

    Mishra, Rakesh K; Dhawan, Jyotsna

    2010-01-01

    The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation. PMID:20592864

  12. Linker DNA destabilizes condensed chromatin.

    PubMed

    Green, G R; Ferlita, R R; Walkenhorst, W F; Poccia, D L

    2001-01-01

    The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

  13. Statistical mechanics of nucleosome ordering by chromatin-structure-induced two-body interactions.

    PubMed

    Chereji, Răzvan V; Tolkunov, Denis; Locke, George; Morozov, Alexandre V

    2011-05-01

    One-dimensional arrays of nucleosomes (DNA-bound histone octamers separated by stretches of linker DNA) fold into higher-order chromatin structures which ultimately make up eukaryotic chromosomes. Chromatin structure formation leads to 10-11 base pair (bp) discretization of linker lengths caused by the smaller free energy cost of packaging nucleosomes into regular chromatin fibers if their rotational setting (defined by the DNA helical twist) is conserved. We describe nucleosome positions along the fiber using a thermodynamic model of finite-size particles with both intrinsic histone-DNA interactions and an effective two-body potential. We infer one- and two-body energies directly from high-throughput maps of nucleosome positions. We show that higher-order chromatin structure helps explains in vitro and in vivo nucleosome ordering in transcribed regions, and plays a leading role in establishing well-known 10-11 bp genome-wide periodicity of nucleosome positions.

  14. Statistical mechanics of nucleosome ordering by chromatin-structure-induced two-body interactions

    NASA Astrophysics Data System (ADS)

    Chereji, Răzvan V.; Tolkunov, Denis; Locke, George; Morozov, Alexandre V.

    2011-05-01

    One-dimensional arrays of nucleosomes (DNA-bound histone octamers separated by stretches of linker DNA) fold into higher-order chromatin structures which ultimately make up eukaryotic chromosomes. Chromatin structure formation leads to 10-11 base pair (bp) discretization of linker lengths caused by the smaller free energy cost of packaging nucleosomes into regular chromatin fibers if their rotational setting (defined by the DNA helical twist) is conserved. We describe nucleosome positions along the fiber using a thermodynamic model of finite-size particles with both intrinsic histone-DNA interactions and an effective two-body potential. We infer one- and two-body energies directly from high-throughput maps of nucleosome positions. We show that higher-order chromatin structure helps explains in vitro and in vivo nucleosome ordering in transcribed regions, and plays a leading role in establishing well-known 10-11 bp genome-wide periodicity of nucleosome positions.

  15. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  16. Chromatin shapes the mitotic spindle.

    PubMed

    Dinarina, Ana; Pugieux, Céline; Corral, Maria Mora; Loose, Martin; Spatz, Joachim; Karsenti, Eric; Nédélec, François

    2009-08-07

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

  17. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Thelen, M P; Noy, A

    2006-02-06

    In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

  18. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

    Genomic DNA is packaged into chromatin in eukaryotic cells. The fundamental building block of chromatin is the nucleosome, a 147 bp-long DNA molecule wrapped around the surface of a histone octamer. Arrays of nucleosomes are positioned along DNA according to their sequence preferences and folded into higher-order chromatin fibers whose structure is poorly understood. We have developed a framework for predicting sequence-specific histone-DNA interactions and the effective two-body potential responsible for ordering nucleosomes into regular higher-order structures. Our approach is based on the analogy between nucleosomal arrays and a one-dimensional fluid of finite-size particles with nearest-neighbor interactions. We derive simple rules which allow us to predict nucleosome occupancy solely from the dinucleotide content of the underlying DNA sequences.Dinucleotide content determines the degree of stiffness of the DNA polymer and thus defines its ability to bend into the nucleosomal superhelix. As expected, the nucleosome positioning rules are universal for chromatin assembled in vitro on genomic DNA from baker's yeast and from the nematode worm C.elegans, where nucleosome placement follows intrinsic sequence preferences and steric exclusion. However, the positioning rules inferred from in vivo C.elegans chromatin are affected by global nucleosome depletion from chromosome arms relative to central domains, likely caused by the attachment of the chromosome arms to the nuclear membrane. Furthermore, intrinsic nucleosome positioning rules are overwritten in transcribed regions, indicating that chromatin organization is actively managed by the transcriptional and splicing machinery.

  19. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

    PubMed Central

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A.; Irudayaraj, Joseph M. K.

    2015-01-01

    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. PMID:25501817

  20. Chromatin Remodeling and Plant Immunity.

    PubMed

    Chen, W; Zhu, Q; Liu, Y; Zhang, Q

    2017-01-01

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance?

  1. Evolution of histone 2A for chromatin compaction in eukaryotes

    PubMed Central

    Macadangdang, Benjamin R; Oberai, Amit; Spektor, Tanya; Campos, Oscar A; Sheng, Fang; Carey, Michael F; Vogelauer, Maria; Kurdistani, Siavash K

    2014-01-01

    During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine–DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer. DOI: http://dx.doi.org/10.7554/eLife.02792.001 PMID:24939988

  2. Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

    PubMed Central

    Holt, Matthew; Muir, Tom

    2016-01-01

    Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments. PMID:25784050

  3. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  4. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

    The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions. PMID:23471416

  5. New insights into chromatin folding and dynamics from multi-scale modeling

    NASA Astrophysics Data System (ADS)

    Olson, Wilma

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of roughly 150 DNA base pairs and eight histone proteins-found on chromatin fibers. We have developed a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs with 3-25 evenly spaced nucleosomes. The correspondence between the predicted and observed effects of nucleosome composition, spacing, and numbers on long-range communication between regulatory proteins bound to the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We have extracted effective nucleosome-nucleosome potentials from the mesoscale simulations and introduced the potentials in a larger scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable influence of nucleosome spacing on chromatin flexibility. Small changes in the length of the DNA fragments linking successive nucleosomes introduce marked changes in the local interactions of the nucleosomes and in the spatial configurations of the fiber as a whole. The changes in nucleosome positioning influence the statistical properties of longer chromatin constructs with 100-10,000 nucleosomes. We are investigating the extent to which the `local' interactions of regularly spaced nucleosomes contribute to the corresponding interactions in chains with mixed spacings as a step toward the treatment of fibers with nucleosomes positioned at the sites mapped at base-pair resolution on genomic sequences. Support of the work by USPHS R01 GM 34809 is gratefully acknowledged.

  6. Atomic force microscopy of chromatin arrays reveal non-monotonic salt dependence of array compaction in solution

    PubMed Central

    Krzemien, Katarzyna M.; Beckers, Maximilian; Quack, Salina; Michaelis, Jens

    2017-01-01

    Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA- protein complexes. Here, we perform high-resolution atomic force microscopy of non-cross-linked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation. PMID:28296908

  7. Chromatin signatures of the Drosophila replication program.

    PubMed

    Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

    2011-02-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

  8. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  9. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  10. HDACi--targets beyond chromatin.

    PubMed

    Buchwald, Marc; Krämer, Oliver H; Heinzel, Thorsten

    2009-08-08

    Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anti-cancer drugs, which induce histone (hyper-) acetylation and counteract aberrant gene repression. On the other hand, HDACi treatment can also result in decreased gene expression, and targeting HDACs affects more than chromatin. Recently, HDACi were shown to evoke non-histone protein acetylation, which can alter signaling networks relevant for tumorgenesis. Furthermore, HDACi can promote the degradation of (proto-) oncoproteins. Here, we summarize these findings and discuss how these substances could be beneficial for the treatment and prevention of human ailments, such as cancer and unbalanced immune functions.

  11. Genome-Wide Views of Chromatin Structure

    PubMed Central

    Rando, Oliver J.; Chang, Howard Y.

    2010-01-01

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation. PMID:19317649

  12. The Chd Family of Chromatin Remodelers

    PubMed Central

    Marfella, Concetta G.A.; Imbalzano, Anthony N.

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic, biochemical, and structural studies demonstrate that Chd proteins are important regulators of transcription and play critical roles during developmental processes. Numerous Chd proteins are also implicated in human disease. PMID:17350655

  13. Nuclear Phosphoinositide Regulation of Chromatin.

    PubMed

    Hamann, Bree L; Blind, Raymond D

    2017-03-03

    Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology. This article is protected by copyright. All rights reserved.

  14. Identification of lamin B–regulated chromatin regions based on chromatin landscapes

    PubMed Central

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-01-01

    Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation. PMID:25995381

  15. Open chromatin reveals the functional maize genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  16. Chromatin roadblocks to reprogramming 50 years on.

    PubMed

    Skene, Peter J; Henikoff, Steven

    2012-10-29

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.

  17. [Chromatin morphology and cytokinesis in pleurocapsalean cyanobacteria].

    PubMed

    Pinevich, A V; Gavrilova, O V; Averina, S G

    2007-01-01

    By means of differential interference contrast (DIC) and fluorescence microscopy, chromatin morphology and cytokinesis have been described in the cyanobacterium Pleurocapsa sp. CALU 1126 capable of multiple fission (multiple reproduction of the mother cell, the macrocyte, with formation of unique reproductive cells, the baeocytes). Two kinds of chromatin behavior have been revealed in the cell cycle: 1) the formation of numerous chromatin areas before their compartmentalization by multiple fission; 2) chromatin condensation in the phase of binary fission, and chromatin decondensation in growth period. The cytokinetic essence of multiple fission has been shown to consist of successive binary fissions of the macrocyte, while in between the mother cells (pre-baeocytes) do not grow.

  18. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  19. Initiation of meiotic recombination in chromatin structure.

    PubMed

    Yamada, Takatomi; Ohta, Kunihiro

    2013-08-01

    Meiotic homologous recombination is markedly activated during meiotic prophase to play central roles in faithful chromosome segregation and conferring genetic diversity to gametes. It is initiated by programmed DNA double-strand breaks (DSBs) by the conserved protein Spo11, and preferentially occurs at discrete sites called hotspots. Since the functions of Spo11 are influenced by both of local chromatin at hotspots and higher-order chromosome structures, formation of meiotic DSBs is under regulation of chromatin structure. Therefore, investigating features and roles of meiotic chromatin is crucial to elucidate the in vivo mechanism of meiotic recombination initiation. Recent progress in genome-wide chromatin analyses tremendously improved our understanding on this point, but many critical questions are left unaddressed. In this review, we summarize current knowledge in the field, and also discuss the future problems that must be solved to understand the role of chromatin structure in meiotic recombination.

  20. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.

    PubMed

    Morris, Stephanie A; Baek, Songjoon; Sung, Myong-Hee; John, Sam; Wiench, Malgorzata; Johnson, Thomas A; Schiltz, R Louis; Hager, Gordon L

    2014-01-01

    ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

  1. Chromatin Dynamics of Circadian Transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

    The molecular circadian clock orchestrates the daily cyclical expression of thousands of genes. Disruption of this transcriptional program leads to a variety of pathologies, including insomnia, depression and metabolic disorders. Circadian rhythms in gene expression rely on specific chromatin transitions which are ultimately coordinated by the molecular clock. As a consequence, a highly plastic and dynamic circadian epigenome can be delineated across different tissues and cell types. Intriguingly, genome topology appears to coordinate cyclic transcription at circadian interactomes, in which circadian genes are in physical contact within the cell nucleus in a time-specific manner. Moreover, the clock machinery shows functional interplays with key metabolic regulators, thereby connecting the circadian epigenome to cellular metabolism. Unraveling the molecular aspects of such interplays is likely to reveal new therapeutic strategies towards the treatment of metabolic disorders. PMID:27014564

  2. Heavy ion-induced lesions in DNA: A theoretical model for the initial induction of DNA strand breaks and chromatin breaks

    SciTech Connect

    Schmidt, J.B.

    1993-01-01

    A theoretical model has been developed and used to calculate yields and spatial distributions of DNA strand breaks resulting from the interactions of heavy ions with chromatin in aqueous systems. The three dimensional spatial distribution of ionizing events has been modeled for charged particles as a function of charge and velocity. Chromatin has been modeled as a 30 nm diameter solenoid of nucleosomal DNA. The Monte Carlo methods used by Chatterjee et al. have been applied to DNA in a chromatin conformation. Refinements to their methods include: a combined treatment of primary and low energy (<2 keV) secondary electron interactions, an improved low energy delta ray model, and the combined simulation of direct energy deposition on the DNA and attack by diffusing hydroxyl radicals. Individual particle tracks are treated independently, which is assumed to be applicable to low fluence irradiations in which multiple particle effects are negligible. Single strand break cross section [open quotes]hooks[close quotes] seen in experiments at very high LET appear to be due to the collapsing radial extent of the track, as predicted in the [open quotes]deep sieve[close quotes] hypothesis proposed by Tobias et al. Spatial distributions of lesions produced by particles have been found to depend on chromatin structure. In the future, heavy ions may be used as a tool to probe the organization of DNA in chromatin. A Neyman A-binomial variation of the [open quotes]cluster model[close quotes] for the distribution of chromatin breaks per irradiated cell has been theoretically tested. The model includes a treatment of the chromatin fragment detection technique's resolution, which places a limitation on the minimum size of fragments which can be detected. The model appears to fit some of the experimental data reasonably well. However, further experimental and theoretical refinements are desirable.

  3. Effect of hyperthermia on replicating chromatin

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.

    1981-10-01

    The extent of heat-induced structural alterations in chromatin containing nascent (pulse-labeled) DNA was assayed using the enzyme micrococcal nuclease. The basic nucleosome structure in nascent and mature chromatin of S-phase cells appeared unaltered for up to 16 hr after exposure to hyperthermic temperatures as high as 48/sup 0/C for 15 min. However, the rate of nuclease digestion of DNA in both nascent and mature chromatin is inhibited following exposure to hyperthermic temperatures. In unheated cells, pulse-labeled nascent DNA matured into mature chromatin structure with a half-time of 2.5 min. The half-time for the maturation of pulse-labeled DNA from nascent into mature chromatin increased in a linear manner as a function of increasing temperature of exposure with constant heating time at temperatures above 43/sup 0/C. Both the reduced nuclease digestibility of nascent DNA and the increased time for chromatin structural changes could be due to the increased protein mass of chromatin following hyperthermia.

  4. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  5. Nucleosome structure in chromatin from heated cells

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.; Winward, R.T.

    1980-12-01

    The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.

  6. reSETting chromatin during transcription elongation

    PubMed Central

    Smolle, Michaela; Workman, Jerry L.; Venkatesh, Swaminathan

    2013-01-01

    Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal. PMID:23257840

  7. Chromatin targeting drugs in cancer and immunity.

    PubMed

    Prinjha, Rab; Tarakhovsky, Alexander

    2013-08-15

    Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.

  8. Optical fiber-based photocathode

    NASA Astrophysics Data System (ADS)

    Cǎsǎndruc, Albert; Bücker, Robert; Kassier, Günther; Miller, R. J. Dwayne

    2016-08-01

    We present the design of a back-illuminated photocathode for electron diffraction experiments based on an optical fiber, and experimental characterization of emitted electron bunches. Excitation light is guided through the fiber into the experimental vacuum chamber, eliminating typical alignment difficulties between the emitter metal and the optical trigger and position instabilities, as well as providing reliable control of the laser spot size and profile. The in-vacuum fiber end is polished and coated with a 30 nm gold (Au) layer on top of 3 nm of chromium (Cr), which emits electrons by means of single-photon photoemission when femtosecond pulses in the near ultraviolet (257 nm) are fed into the fiber on the air side. The emission area can be adjusted to any value between a few nanometers (using tapered fibers) and the size of a multi-mode fiber core (100 μm or larger). In this proof-of-principle experiment, two different types of fibers were tested, with emission spot diameters of 50 μm and 100 μm, respectively. The normalized thermal electron beam emittance (TE) was measured by means of the aperture scan technique, and a TE of 4.0 π nm was measured for the smaller spot diameter. Straightforward enhancements to the concept allowed to demonstrate operation in an electric field environment of up to 7 MV/m.

  9. Chromatin dynamics during DNA replication

    PubMed Central

    Bar-Ziv, Raz; Voichek, Yoav; Barkai, Naama

    2016-01-01

    Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by ∼5–6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification. PMID:27225843

  10. Centromeric chromatin in fission yeast.

    PubMed

    Partridge, Janet F

    2008-05-01

    A fundamental requirement for life is the ability of cells to divide properly and to pass on to their daughters a full complement of genetic material. The centromere of the chromosome is essential for this process, as it provides the DNA sequences on which the kinetochore (the proteinaceous structure that links centromeric DNA to the spindle microtubules) assembles to allow segregation of the chromosomes during mitosis. It has long been recognized that kinetochore assembly is subject to epigenetic control, and deciphering how centromeres promote faithful chromosome segregation provides a fascinating intellectual challenge. This challenge is made more difficult by the scale and complexity of DNA sequences in metazoan centromeres, thus much research has focused on dissecting centromere function in the single celled eukaryotic yeasts. Interestingly, in spite of similarities in the genome size of budding and fission yeasts, they seem to have adopted some striking differences in their strategy for passing on their chromosomes. Budding yeast have "point" centromeres, where a 125 base sequence is sufficient for mitotic propagation, whereas fission yeast centromeres are more reminiscent of the large repetitive centromeres of metazoans. In addition, the centromeric heterochromatin which coats centromeric domains of fission yeast and metazoan centromeres and is critical for their function, is largely absent from budding yeast centromeres. This review focuses on the assembly and maintenance of centromeric chromatin in the fission yeast.

  11. Transcription of nucleosomes from human chromatin.

    PubMed Central

    Shaw, P A; Sahasrabuddhe, C G; Hodo, H G; Saunders, G F

    1978-01-01

    Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes. PMID:693325

  12. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    di Pierro, Miichele; Zhang, Bin; Wolynes, Peter J.; Onuchic, Jose N.

    In vivo, the human genome folds into well-determined and conserved three-dimensional structures. The mechanism driving the folding process remains unknown. We report a theoretical model (MiChroM) for chromatin derived by using the maximum entropy principle. The proposed model allows Molecular Dynamics simulations of the genome using as input the classification of loci into chromatin types and the presence of binding sites of loop forming protein CTCF. The model was trained to reproduce the Hi-C map of chromosome 10 of human lymphoblastoid cells. With no additional tuning the model was able to predict accurately the Hi-C maps of chromosomes 1-22 for the same cell line. Simulations show unknotted chromosomes, phase separation of chromatin types and a preference of chromatin of type A to sit at the periphery of the chromosomes.

  13. Nucleosome repeat lengths and columnar chromatin structure.

    PubMed

    Trifonov, Edward N

    2016-06-01

    Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.

  14. Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

    PubMed Central

    Martini, Emmanuelle; Roche, Danièle M.J.; Marheineke, Kathrin; Verreault, Alain; Almouzni, Geneviève

    1998-01-01

    The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair. PMID:9813080

  15. Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation▿ †

    PubMed Central

    Das, Chandrima; Hizume, Kohji; Batta, Kiran; Kumar, B. R. Prashanth; Gadad, Shrikanth S.; Ganguly, Semanti; Lorain, Stephanie; Verreault, Alain; Sadhale, Parag P.; Takeyasu, Kunio; Kundu, Tapas K.

    2006-01-01

    Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization. PMID:16982701

  16. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

  17. Links between genome replication and chromatin landscapes.

    PubMed

    Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2015-07-01

    Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication.

  18. Programming smooth muscle plasticity with chromatin dynamics.

    PubMed

    McDonald, Oliver G; Owens, Gary K

    2007-05-25

    Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues. For example, vascular SMCs undergo profound changes in their phenotype during neointimal formation in response to vessel injury or within atherosclerotic plaques. Recent studies have shown that interaction of serum response factor (SRF) and its numerous accessory cofactors with CArG box DNA sequences within promoter chromatin of SMC genes is a nexus for integrating signals that influence SMC differentiation in development and disease. During development, SMC-restricted sets of posttranslational histone modifications are acquired within the CArG box chromatin of SMC genes. These modifications in turn control the chromatin-binding properties of SRF. The histone modifications appear to encode a SMC-specific epigenetic program that is used by extracellular cues to influence SMC differentiation, by regulating binding of SRF and its partners to the chromatin template. Thus, SMC differentiation is dynamically regulated by the interplay between SRF accessory cofactors, the SRF-CArG interaction, and the underlying histone modification program. As such, the inherent plasticity of the SMC lineage offers unique glimpses into how cellular differentiation is dynamically controlled at the level of chromatin within the context of changing microenvironments. Further elucidation of how chromatin regulates SMC differentiation will undoubtedly yield valuable insights into both normal developmental processes and the pathogenesis of several vascular diseases that display detrimental SMC phenotypic behavior.

  19. Chromatin compaction in terminally differentiated avian blood cells: the role of linker histone H5 and non-histone protein MENT.

    PubMed

    Kowalski, Andrzej; Pałyga, Jan

    2011-07-01

    Chromatin has a tendency to shift from a relatively decondensed (active) to condensed (inactive) state during cell differentiation due to interactions of specific architectural and/or regulatory proteins with DNA. A promotion of chromatin folding in terminally differentiated avian blood cells requires the presence of either histone H5 in erythrocytes or non-histone protein, myeloid and erythroid nuclear termination stage-specific protein (MENT), in white blood cells (lymphocytes and granulocytes). These highly abundant proteins assist in folding of nucleosome arrays and self-association of chromatin fibers into compacted chromatin structures. Here, we briefly review structural aspects and molecular mode of action by which these unrelated proteins can spread condensed chromatin to form inactivated regions in the genome.

  20. Histone variants: the tricksters of the chromatin world☆

    PubMed Central

    Volle, Catherine; Dalal, Yamini

    2014-01-01

    The eukaryotic genome exists in vivo at an equimolar ratio with histones, thus forming a polymer composed of DNA and histone proteins. Each nucleosomal unit in this polymer provides versatile capabilities and dynamic range. Substitutions of the individual components of the histone core with structurally distinct histone variants and covalent modifications alter the local fabric of the chromatin fiber, resulting in epigenetic changes that can be regulated by the cell. In this review, we highlight recent advances in the study of histone variant structure, assembly, and inheritance, their influence on nucleosome positioning, and their cumulative effect upon gene expression, DNA repair and the progression of disease. We also highlight fundamental questions that remain unanswered regarding the behavior of histone variants and their influence on cellular function in the normal and diseased states. PMID:24463272

  1. The Dynamics of Individual Nucleosomes Controls the Chromatin Condensation Pathway: Direct Atomic Force Microscopy Visualization of Variant Chromatin

    PubMed Central

    Montel, Fabien; Menoni, Hervé; Castelnovo, Martin; Bednar, Jan; Dimitrov, Stefan; Angelov, Dimitar; Faivre-Moskalenko, Cendrine

    2009-01-01

    Abstract Chromatin organization and dynamics is studied at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique, and numerical modeling. We show that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by atomic force microscopy, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show “beads on a string” structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results suggest strongly that higher-order organization of H1-free nucleosomal arrays is determined mainly by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers. PMID:19619469

  2. A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2011-02-01

    The focus of this review is the dual functions of the sperm chromatin stabilization and how external factors can interfere with these functions. Zinc depletion after ejaculation allows for rapid and total sperm chromatin decondensation without addition of exogenous disulfide cleaving agents. Zinc depletion without concomitant repulsion of chromatin fibers induces another type of stability that requires exogenous disulfide cleaving agents to allow decondensation. It is essential to extend the present concept, that the sperm chromatin stability is based on disulfide bridges only, to include also the functions of Zn(2+). It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn(2+) that stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism: the formation of zinc bridges involving protamine thiols of cysteine and potentially also imidazole groups of histidine. Extraction of zinc from the freshly ejaculated spermatozoon allows two totally different biological results: (1) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g., through phosphorylation in the ooplasm) and (2) thiols freed from Zn(2+) are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (in first ejaculated fraction) represent physiology. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid, a situation common to most assisted reproductive techniques (ART) laboratories where the entire ejaculate is collected into a single container in which spermatozoa and secretions are mixed during at least 30 min. Some men in infertile couples have low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to

  3. Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1

    PubMed Central

    Conn, Kristen L.; Schang, Luis M.

    2013-01-01

    Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells. PMID:23863878

  4. A computer lab exploring evolutionary aspects of chromatin structure and dynamics for an undergraduate chromatin course*.

    PubMed

    Eirín-López, José M

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a vast body of knowledge to the classroom. With this aim, the present work describes a multidisciplinary computer lab designed to introduce undergraduate students to the dynamic nature of chromatin, within the context of the one semester course "Chromatin: Structure, Function and Evolution." This exercise is organized in three parts including (a) molecular evolutionary biology of histone families (using the H1 family as example), (b) histone structure and variation across different animal groups, and (c) effect of histone diversity on nucleosome structure and chromatin dynamics. By using freely available bioinformatic tools that can be run on common computers, the concept of chromatin dynamics is interactively illustrated from a comparative/evolutionary perspective. At the end of this computer lab, students are able to translate the bioinformatic information into a biochemical context in which the relevance of histone primary structure on chromatin dynamics is exposed. During the last 8 years this exercise has proven to be a powerful approach for teaching chromatin structure and dynamics, allowing students a higher degree of independence during the processes of learning and self-assessment.

  5. A Computer Lab Exploring Evolutionary Aspects of Chromatin Structure and Dynamics for an Undergraduate Chromatin Course

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a…

  6. Elucidate Chromatin Folding at the Mesoscale

    NASA Astrophysics Data System (ADS)

    Qiu, Xiangyun

    Knowledge of the three-dimensional structure of chromatin, an active participant of all gene-directed processes, is required to decode its (epi)genetics-structure-function relationships. Albeit often simplified as ``beads-on-a-string'', chromatin possesses daunting complexity in its intricate intra- and inter-nucleosome interactions, as well as the myriad types of molecules acting on it. On the other hand, the folding of chromatin from an extended chain of nucleosomes is highly constrained, e.g., by rather bulky nucleosomes and semi-rigid linker dsDNAs. Further given the well-defined nucleosome and dsDNA structures at the nanometer scale, this creates an opportunity for low-resolution structural methods such as small angle scattering to obtain mesoscale structures of chromatin, which can be further refined computationally to yield atomistic structures of chromatin. Here we present results from our recent studies of recombinant nucleosome arrays with solution small angle x-ray scattering (SAXS) and ensemble structure modeling.

  7. Toxicants and human sperm chromatin integrity.

    PubMed

    Delbès, Geraldine; Hales, Barbara F; Robaire, Bernard

    2010-01-01

    The integrity of the paternal genome is essential as the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome. During the past two decades, many assays have been developed to measure sperm DNA strand breaks, chromatin structure and compaction and assess the proteins associated with the DNA, as well as epigenetic modifications. Using these assays, it has been shown that exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, can affect the integrity of sperm chromatin, inducing structural, genetic and/or epigenetic abnormalities. The mechanisms by which such damage is triggered are still largely unresolved and the susceptibility of each individual will depend on their genetic background, lifestyle and exposure to various insults. Depending on the nature of the chemicals, they may directly target the DNA, induce an oxidative stress, or modify the epigenetic elements. The significance of measuring the sperm chromatin integrity comes from the fact that this end-point correlates well with the low IVF and ICSI outcomes, and idiopathic infertility. Nevertheless, it is hard to establish a direct link between the paternal sperm chromatin integrity and the health of the future generations. Thus, it seems essential to undertake studies that will resolve the impact of chemical and environmental factors on chromatin structure and epigenetic components of human spermatozoa and to elucidate what sperm nuclear end-points are predictors of the quality of progeny outcome.

  8. Chromatin Proteins: Key Responders to Stress

    PubMed Central

    Smith, Karen T.; Workman, Jerry L.

    2012-01-01

    Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress. PMID:22859908

  9. Reshaping chromatin after DNA damage: the choreography of histone proteins.

    PubMed

    Polo, Sophie E

    2015-02-13

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.

  10. Predictive chromatin signatures in the mammalian genome

    PubMed Central

    Hon, Gary C.; Hawkins, R. David; Ren, Bing

    2009-01-01

    The DNA sequence of an organism is a blueprint of life: it harbors not only the information about proteins and other molecules produced in each cell, but also instructions on when and where such molecules are made. Chromatin, the structure of histone and DNA that has co-evolved with eukaryotic genome, also contains information that indicates the function and activity of the underlying DNA sequences. Such information exists in the form of covalent modifications to the histone proteins that comprise the nucleosome. Thanks to the development of high throughput technologies such as DNA microarrays and next generation DNA sequencing, we have begun to associate the various combinations of chromatin modification patterns with functional sequences in the human genome. Here, we review the rapid progress from descriptive observations of histone modification profiles to highly predictive models enabling use of chromatin signatures to enumerate novel functional sequences in mammalian genomes that have escaped previous detection. PMID:19808796

  11. Nucleosome dynamics during chromatin remodeling in vivo.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation.

  12. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  13. Open chromatin reveals the functional maize genome

    PubMed Central

    Rodgers-Melnick, Eli; Vera, Daniel L.; Bass, Hank W.

    2016-01-01

    Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome. PMID:27185945

  14. Chromatin Higher-order Structure and Dynamics

    PubMed Central

    Woodcock, Christopher L.; Ghosh, Rajarshi P.

    2010-01-01

    The primary role of the nucleus as an information storage, retrieval, and replication site requires the physical organization and compaction of meters of DNA. Although it has been clear for many years that nucleosomes constitute the first level of chromatin compaction, this contributes a relatively small fraction of the condensation needed to fit the typical genome into an interphase nucleus or set of metaphase chromosomes, indicating that there are additional “higher order” levels of chromatin condensation. Identifying these levels, their interrelationships, and the principles that govern their occurrence has been a challenging and much discussed problem. In this article, we focus on recent experimental advances and the emerging evidence indicating that structural plasticity and chromatin dynamics play dominant roles in genome organization. We also discuss novel approaches likely to yield important insights in the near future, and suggest research areas that merit further study. PMID:20452954

  15. 4D chromatin dynamics in cycling cells

    PubMed Central

    Strickfaden, Hilmar; Zunhammer, Andreas; van Koningsbruggen, Silvana; Köhler, Daniela

    2010-01-01

    This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements. PMID:21327076

  16. CHD chromatin remodelers and the transcription cycle.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by "opening" or "closing" chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

  17. Spindle assembly on immobilized chromatin micropatterns.

    PubMed

    Pugieux, Céline; Dmitrieff, Serge; Tarnawska, Katarzyna; Nédélec, François

    2014-01-01

    We describe a method to assemble meiotic spindles on immobilized micropatterns of chromatin built on a first layer of biotinylated BSA deposited by microcontact printing. Such chromatin patterns routinely produce bipolar spindles with a yield of 60%, and offer the possibility to follow spindle assembly dynamics, from the onset of nucleation to the establishment of a quasi steady state. Hundreds of spindles can be recorded in parallel for different experimental conditions. We also describe the semi-automated image analysis pipeline, which is used to analyze the assembly kinetics of spindle arrays, or the final morphological diversity of the spindles.

  18. Dynamics of Histone Tails within Chromatin

    NASA Astrophysics Data System (ADS)

    Bernier, Morgan; North, Justin; Page, Michael; Jaroniec, Christopher; Hammel, Christopher; Poirier, Michael

    2012-02-01

    Genetic information in humans is encoded within DNA molecules that is wrapped around histone octamer proteins and compacted into a highly conserved structural polymer, chromatin. The physical and material properties of chromatin appear to influence gene expression by altering the accessibility of proteins to the DNA. The tails of the histones are flexible domains that are thought to play a role in regulating DNA accessibility and compaction; however the molecular mechanisms for these phenomena are not understood. I will present CW-EPR studies on site directed spin labeled nucleosomes that probe the structure and dynamics of these histone tails within nucleosomes.

  19. Allele-specific chromatin immunoprecipitation studies show genetic influence on chromatin state in human genome.

    PubMed

    Kadota, Mitsutaka; Yang, Howard H; Hu, Nan; Wang, Chaoyu; Hu, Ying; Taylor, Philip R; Buetow, Kenneth H; Lee, Maxwell P

    2007-05-18

    Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena.

  20. Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length

    SciTech Connect

    Williams, S.P.; Langmore, J.P. )

    1991-03-01

    Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 {plus minus} 0.5, 12.1 {plus minus} 0.4, and 15.9 {plus minus} 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 {plus minus} 0.5, 8.3 {plus minus} 0.6, and 11.8 {plus minus} 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.

  1. Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

    PubMed

    Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar

    2016-12-20

    Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin?

  2. Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter

    PubMed Central

    Müller, Waltraud G.; Walker, Dawn; Hager, Gordon L.; McNally, James G.

    2001-01-01

    We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a ∼0.5-μm spot to form a fiber 1–10 μm long. Arrays later recondensed by 3–8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and α-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously. PMID:11448988

  3. The great repression: chromatin and cryptic transcription.

    PubMed

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity.

  4. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  5. An Isochore Framework Underlies Chromatin Architecture

    PubMed Central

    Jabbari, Kamel; Bernardi, Giorgio

    2017-01-01

    A recent investigation showed the existence of correlations between the architectural features of mammalian interphase chromosomes and the compositional properties of isochores. This result prompted us to compare maps of the Topologically Associating Domains (TADs) and of the Lamina Associated Domains (LADs) with the corresponding isochore maps of mouse and human chromosomes. This approach revealed that: 1) TADs and LADs correspond to isochores, i.e., isochores are the genomic units that underlie chromatin domains; 2) the conservation of TADs and LADs in mammalian genomes is explained by the evolutionary conservation of isochores; 3) chromatin domains corresponding to GC-poor isochores (e.g., LADs) show not only self-interactions but also intrachromosomal interactions with other domains also corresponding to GC-poor isochores even if located far away; in contrast, chromatin domains corresponding to GC-rich isochores (e.g., TADs) show more localized chromosomal interactions, many of which are inter-chromosomal. In conclusion, this investigation establishes a link between DNA sequences and chromatin architecture, explains the evolutionary conservation of TADs and LADs and provides new information on the spatial distribution of GC-poor/gene-poor and GC-rich/gene-rich chromosomal regions in the interphase nucleus. PMID:28060840

  6. Chemical biology: Chromatin chemistry goes cellular

    NASA Astrophysics Data System (ADS)

    Fischle, Wolfgang; Schwarzer, Dirk; Mootz, Henning D.

    2015-05-01

    Analysing post-translational modifications of histone proteins as they occur within chromatin is challenging due to their large number and chemical diversity. A major step forward has now been achieved by using split intein chemistry to engineer functionalized histones within cells.

  7. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.

  8. Inferential modeling of 3D chromatin structure.

    PubMed

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-04-30

    For eukaryotic cells, the biological processes involving regulatory DNA elements play an important role in cell cycle. Understanding 3D spatial arrangements of chromosomes and revealing long-range chromatin interactions are critical to decipher these biological processes. In recent years, chromosome conformation capture (3C) related techniques have been developed to measure the interaction frequencies between long-range genome loci, which have provided a great opportunity to decode the 3D organization of the genome. In this paper, we develop a new Bayesian framework to derive the 3D architecture of a chromosome from 3C-based data. By modeling each chromosome as a polymer chain, we define the conformational energy based on our current knowledge on polymer physics and use it as prior information in the Bayesian framework. We also propose an expectation-maximization (EM) based algorithm to estimate the unknown parameters of the Bayesian model and infer an ensemble of chromatin structures based on interaction frequency data. We have validated our Bayesian inference approach through cross-validation and verified the computed chromatin conformations using the geometric constraints derived from fluorescence in situ hybridization (FISH) experiments. We have further confirmed the inferred chromatin structures using the known genetic interactions derived from other studies in the literature. Our test results have indicated that our Bayesian framework can compute an accurate ensemble of 3D chromatin conformations that best interpret the distance constraints derived from 3C-based data and also agree with other sources of geometric constraints derived from experimental evidence in the previous studies. The source code of our approach can be found in https://github.com/wangsy11/InfMod3DGen.

  9. The many faces of plant chromatin: Meeting summary of the 4th European workshop on plant chromatin 2015, Uppsala, Sweden.

    PubMed

    Mozgová, Iva; Köhler, Claudia; Gaudin, Valérie; Hennig, Lars

    2015-01-01

    In June 2015, the fourth European Workshop on Plant Chromatin took place in Uppsala, Sweden, bringing together 80 researchers studying various aspects of plant chromatin and epigenetics. The intricate relationships between plant chromatin dynamics and gene expression change, chromatin organization within the plant cell nucleus, and the impact of chromatin structure on plant development were discussed. Among the main highlights of the meeting were an ever-growing list of newly identified players in chromatin structure establishment and the development of novel tools and approaches to foster our understanding of chromatin-mediated gene regulation, taking into account the context of the plant cell nucleus and its architecture. In this report, we summarize some of the main advances and prospects of plant chromatin research presented at this meeting.

  10. The many faces of plant chromatin: Meeting summary of the 4th European workshop on plant chromatin 2015, Uppsala, Sweden

    PubMed Central

    Mozgová, Iva; Köhler, Claudia; Gaudin, Valérie; Hennig, Lars

    2015-01-01

    In June 2015, the fourth European Workshop on Plant Chromatin took place in Uppsala, Sweden, bringing together 80 researchers studying various aspects of plant chromatin and epigenetics. The intricate relationships between plant chromatin dynamics and gene expression change, chromatin organization within the plant cell nucleus, and the impact of chromatin structure on plant development were discussed. Among the main highlights of the meeting were an ever-growing list of newly identified players in chromatin structure establishment and the development of novel tools and approaches to foster our understanding of chromatin-mediated gene regulation, taking into account the context of the plant cell nucleus and its architecture. In this report, we summarize some of the main advances and prospects of plant chromatin research presented at this meeting. PMID:26646904

  11. The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

    PubMed Central

    Sarkar, Sovan; Kiely, Rhian

    2010-01-01

    Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair. PMID:21135142

  12. Chromatin opening of DNA satellites by targeted sequence-specific drugs.

    PubMed

    Janssen, S; Durussel, T; Laemmli, U K

    2000-11-01

    There are few tools available for dissecting and elucidating the functions of DNA satellites and other nongenic DNA. To address this, we have explored the experimental potential of DNA sequence-specific drugs containing pyrrole and imidazole amino acids (polyamides). Compounds were synthesized that target different Drosophila melanogaster satellites. Dimeric oligopyrroles were shown to target the AT-rich satellites I, III, and SARs (scaffold associated regions). One polyamide (P31) specifically binds the GAGAA satellite V. Specificity of targeting was established by footprinting, epifluorescence of nuclei, and polytene chromosomes stained with fluorescent derivatives. These polyamides were shown to mediate satellite-specific chromatin opening of the chromatin fiber. Remarkably, certain polyamides induced defined gain or loss-of-function phenotypes when fed to Drosophila melanogaster.

  13. Large Scale Chromosome Folding Is Stable against Local Changes in Chromatin Structure

    PubMed Central

    Therizols, Pierre

    2016-01-01

    Characterizing the link between small-scale chromatin structure and large-scale chromosome folding during interphase is a prerequisite for understanding transcription. Yet, this link remains poorly investigated. Here, we introduce a simple biophysical model where interphase chromosomes are described in terms of the folding of chromatin sequences composed of alternating blocks of fibers with different thicknesses and flexibilities, and we use it to study the influence of sequence disorder on chromosome behaviors in space and time. By employing extensive computer simulations, we thus demonstrate that chromosomes undergo noticeable conformational changes only on length-scales smaller than 105 basepairs and time-scales shorter than a few seconds, and we suggest there might exist effective upper bounds to the detection of chromosome reorganization in eukaryotes. We prove the relevance of our framework by modeling recent experimental FISH data on murine chromosomes. PMID:27295501

  14. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

    Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J

    2016-07-05

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

  15. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  16. Quantification of chromatin condensation level by image processing.

    PubMed

    Irianto, Jerome; Lee, David A; Knight, Martin M

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation.

  17. Solenoidal model for superstructure in chromatin.

    PubMed Central

    Finch, J T; Klug, A

    1976-01-01

    Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei. Images PMID:1064861

  18. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

    The plant life cycle alternates between a diploid sporophytic phase and haploid gametophytic phase, with the latter giving rise to the gametes. Male gametophyte development encompasses two mitotic divisions that results in a simple three-celled structure knows as the pollen grain, in which two sperm cells are encased within a larger vegetative cell. Both cell types exhibit a very different type of chromatin organization - highly condensed in sperm cell nuclei and highly diffuse in the vegetative cell. Distinct classes of histone variants have dynamic and differential expression in the two cell lineages of the male gametophyte. Here we review how the dynamics of histone variants are linked to reprogramming of chromatin activities in the male gametophyte, compaction of the sperm cell genome and zygotic transitions post-fertilization.

  19. Transcription, chromatin condensation, and gene migration

    PubMed Central

    2009-01-01

    The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in understanding nuclear organization and function. Through the use of more natural tandem DNA arrays, Hu et al. (Hu, Y., I. Kireev, M. Plutz, N. Ashourian, and A.S. Belmont. 2009. J. Cell Biol. 185:87–100) gain new insights into chromatin organization and dynamics, and into the association of splicing factors with active genes. PMID:19349577

  20. Reproducibility of 3D chromatin configuration reconstructions

    PubMed Central

    Segal, Mark R.; Xiong, Hao; Capurso, Daniel; Vazquez, Mariel; Arsuaga, Javier

    2014-01-01

    It is widely recognized that the three-dimensional (3D) architecture of eukaryotic chromatin plays an important role in processes such as gene regulation and cancer-driving gene fusions. Observing or inferring this 3D structure at even modest resolutions had been problematic, since genomes are highly condensed and traditional assays are coarse. However, recently devised high-throughput molecular techniques have changed this situation. Notably, the development of a suite of chromatin conformation capture (CCC) assays has enabled elicitation of contacts—spatially close chromosomal loci—which have provided insights into chromatin architecture. Most analysis of CCC data has focused on the contact level, with less effort directed toward obtaining 3D reconstructions and evaluating the accuracy and reproducibility thereof. While questions of accuracy must be addressed experimentally, questions of reproducibility can be addressed statistically—the purpose of this paper. We use a constrained optimization technique to reconstruct chromatin configurations for a number of closely related yeast datasets and assess reproducibility using four metrics that measure the distance between 3D configurations. The first of these, Procrustes fitting, measures configuration closeness after applying reflection, rotation, translation, and scaling-based alignment of the structures. The others base comparisons on the within-configuration inter-point distance matrix. Inferential results for these metrics rely on suitable permutation approaches. Results indicate that distance matrix-based approaches are preferable to Procrustes analysis, not because of the metrics per se but rather on account of the ability to customize permutation schemes to handle within-chromosome contiguity. It has recently been emphasized that the use of constrained optimization approaches to 3D architecture reconstruction are prone to being trapped in local minima. Our methods of reproducibility assessment provide a

  1. Dynamic chromatin regulation at Notch target genes

    PubMed Central

    Giaimo, Benedetto Daniele; Oswald, Franz; Borggrefe, Tilman

    2017-01-01

    ABSTRACT RBPJ is the central transcription factor that controls the Notch-dependent transcriptional response by coordinating repressing histone H3K27 deacetylation and activating histone H3K4 methylation. Here, we discuss the molecular mechanisms how RBPJ interacts with opposing NCoR/HDAC-corepressing or KMT2D/UTX-coactivating complexes and how this is controlled by phosphorylation of chromatin modifiers. PMID:28027012

  2. [The biological aspects of chromatin diminution].

    PubMed

    Akif'ev, A P; Grishanin, A K

    1993-01-01

    The chromatine diminution (CD), first discovered by Boveri (1887) in ascarids, represents programmed elimination of a part of genetic material in the nuclei of the somatic cells in cyclops and ascarids, and in the protist macronuclei. The CD can be considered as a macromutation sharply changing chromosomal structure, though minimally effecting the phenotype. The analysis of CD is of significance for discussing mechanisms of origin of chromosomal organization, transformation of genome molecular structure in eucaryote evolution, role of the extra DNA.

  3. Premature chromatin condensation upon accumulation of NIMA.

    PubMed Central

    O'Connell, M J; Norbury, C; Nurse, P

    1994-01-01

    The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis. Images PMID:7957060

  4. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  5. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

    PubMed Central

    Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2015-01-01

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression. PMID:26457678

  6. Plant chromatin warms up in Madrid: meeting summary of the 3rd European Workshop on Plant Chromatin 2013, Madrid, Spain.

    PubMed

    Jarillo, José A; Gaudin, Valérie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

    2014-04-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC.

  7. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    PubMed Central

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  8. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility.

    PubMed

    Lamparter, David; Marbach, Daniel; Rueedi, Rico; Bergmann, Sven; Kutalik, Zoltán

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors.

  9. Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

    PubMed Central

    Smolle, Michaela; Venkatesh, Swaminathan; Gogol, Madelaine M.; Li, Hua; Zhang, Ying; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, yet plays a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions and thus maintains chromatin integrity during transcription elongation by RNA polymerase II. PMID:22922743

  10. Capture of associated targets on chromatin links long-distance chromatin looping to transcriptional coordination

    PubMed Central

    Bourgo, Ryan J.; Singhal, Hari; Greene, Geoffrey L.

    2016-01-01

    Here we describe a sensitive and novel method of identifying endogenous DNA–DNA interactions. Capture of Associated Targets on CHromatin (CATCH) uses efficient capture and enrichment of specific genomic loci of interest through hybridization and subsequent purification via complementary biotinylated oligonucleotide. The CATCH assay requires no enzymatic digestion or ligation, requires little starting material, provides high-quality data, has excellent reproducibility and is completed in less than 24 h. Efficacy is demonstrated through capture of three disparate loci, which demonstrate unique subsets of long-distance chromatin interactions enriched for both enhancer marks and oestrogen receptor-binding sites. In each experiment, CATCH-seq peaks representing long-distance chromatin interactions were centred near the TSS of genes, and, critically, the genes identified as physically interacting are shown to be transcriptionally coexpressed. These interactions could potentially create transcriptional hubs for the regulation of gene expression programmes. PMID:27634217

  11. In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

    PubMed Central

    Moné, Martijn J.; Bernas, Tytus; Dinant, Christoffel; Goedvree, Feliks A.; Manders, Erik M. M.; Volker, Marcel; Houtsmuller, Adriaan B.; Hoeijmakers, Jan H. J.; Vermeulen, Wim; van Driel, Roel

    2004-01-01

    Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of ≈30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells. PMID:15520397

  12. Drugging Chromatin in Cancer: Recent Advances and Novel Approaches

    PubMed Central

    Cai, Sheng F.; Chen, Chun-Wei; Armstrong, Scott A.

    2015-01-01

    Chromatin regulatory mechanisms play a major role in the control of gene expression programs during normal development and are disrupted in specific disease states, particularly in cancer. Important mediators of chromatin regulatory processes can broadly be classified into writers, erasers, and readers of covalent chromatin modifications that modulate eukaryotic gene transcription and maintain the integrity of the genome. The reversibility and disease-specific nature of these chromatin states make these regulators attractive therapeutic targets. As such, there is an ever-increasing number of candidate therapies aimed at targeting cancer-associated chromatin states that are in various stages of preclinical and clinical development. In this review, we discuss recent advances that have been made in the rational therapeutic targeting of chromatin regulatory mechanisms and highlight certain cancers where there is a specific rationale to assess these therapeutic approaches. PMID:26590715

  13. Roles of chromatin remodellers in DNA double strand break repair.

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

    Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

  14. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

    Zhang, Pingyu; Torres, Keila; Liu, Xiuping; Liu, Chang-gong; Pollock, Raphael E.

    2016-01-01

    In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatin- regulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate–ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells. PMID:26796306

  15. In vitro Chromatin Assembly - Strategies and Quality Control

    PubMed Central

    Muthurajan, Uma; Mattiroli, Francesca; Bergeron, Serge; Zhou, Keda; Gu, Yajie; Chakravarthy, Srinivas; Dyer, Pamela; Irving, Thomas; Luger, Karolin

    2016-01-01

    Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates. PMID:27372747

  16. A role for chromatin topology in imprinted domain regulation.

    PubMed

    MacDonald, William A; Sachani, Saqib S; White, Carlee R; Mann, Mellissa R W

    2016-02-01

    Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

  17. Aging by epigenetics-A consequence of chromatin damage?

    SciTech Connect

    Sedivy, John M. Banumathy, Gowrishankar; Adams, Peter D.

    2008-06-10

    Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.

  18. NET23/STING promotes chromatin compaction from the nuclear envelope.

    PubMed

    Malik, Poonam; Zuleger, Nikolaj; de las Heras, Jose I; Saiz-Ros, Natalia; Makarov, Alexandr A; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A; Schirmer, Eric C

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

  19. NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    PubMed Central

    de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  20. Histone chaperone networks shaping chromatin function.

    PubMed

    Hammond, Colin M; Strømme, Caroline B; Huang, Hongda; Patel, Dinshaw J; Groth, Anja

    2017-03-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

  1. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  2. Mechanisms of ATP Dependent Chromatin Remodeling

    PubMed Central

    Gangaraju, Vamsi K.; Bartholomew, Blaine

    2007-01-01

    The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed. PMID:17306844

  3. Radiation-induced thymine base damage in replicating chromatin

    SciTech Connect

    Warters, R.L.; Childers, T.J.

    1982-06-01

    The efficiency of radiation-induced production of 5',6'-dihydroxydihydrothymine (t/sup ..gamma../)-type damage was determined in nascent and mature chromatin DNA for the dose range of 50 to 150 krad. These large doses affected neither the total fraction of nuclear DNA in chromatin subunits nor the nucleosome subunit repeat length. The DNA in nascent chromatin, however, was found to be 3.3 times more sensitive than mature chromatin DNA to ..gamma..-ray (/sup 137/Cs)-induced t/sup ..gamma../-type damage, while thymine damage of this type was uniformly distributed in the nucleosomal DNA of mature chromatin (i.e., in the nucleosome core and spacer DNA). The half-time for the transition of nascent DNA sensitivity to mature chromatin DNA sensitivity levels was the same as the half-time at 37/sup 0/C for the maturation of nascent into mature chromatin structure. The rate at which nascent chromatin matured was unaffected by radiation doses as large as 150 krad. The most logical explanation for the greater sensitivity of nascent DNA to radiation is the decreased concentration of histone chromosomal proteins in nascent chromatin.

  4. Regulation of chromatin structure in the cardiovascular system.

    PubMed

    Rosa-Garrido, Manuel; Karbassi, Elaheh; Monte, Emma; Vondriska, Thomas M

    2013-01-01

    It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. 

  5. Chromatin organization and dynamics in double-strand break repair.

    PubMed

    Seeber, Andrew; Gasser, Susan M

    2016-10-31

    Chromatin is organized and segmented into a landscape of domains that serve multiple purposes. In contrast to transcription, which is controlled by defined sequences at distinct sites, DNA damage can occur anywhere. Repair accordingly must occur everywhere, yet it is inevitably affected by its chromatin environment. In this review, we summarize recent work investigating how changes in chromatin organization facilitate and/or guide DNA double-strand break repair. In addition, we examine new live cell studies on the dynamics of chromatin and the mechanisms that regulate its movement.

  6. DNA content, chromatin supraorganization, nuclear glycoproteins and RNA amounts in hepatocytes of mice expressing insulin-dependent diabetes.

    PubMed

    Mello, Maria Luiza S; Aldrovani, Marcela; Moraes, Alberto Silva; Guaraldo, Ana Maria Aparecida; Vidal, Benedicto de Campos

    2009-01-01

    Chromatin supraorganization and extensibility and nuclear glycoprotein content have been reported to change in hepatocytes from mice during development and aging, as well as under starvation and refeeding conditions. In non-obese diabetic (NOD) mice, the expression of insulin-dependent diabetes may be accompanied by metabolic changes in the liver. These changes are likely to be similar to those involved in the aging processes of non-diabetic animals. Therefore, we hypothesized that the chromatin organization, as well as the physical properties and compositions of hepatocyte nuclei would also be affected in NOD mice in the same way as those in aged non-diabetic mice. Nuclear image parameters were evaluated by image analysis of Feulgen-stained preparations. Chromatin extensibility in response to gravity was observed with polarized light after lysis and toluidine blue staining. The Con-A response of nuclear glycoproteins was evaluated with scanning microspectrophotometry. These characteristics were assessed using hepatocyte imprints from female NOD mice after a 28-day period of diabetes expression. Observations and measurements were made in comparison to healthy BALB/c mice. Total RNA amounts were determined for livers of NOD and BALB/c mice. Enhanced polyploidy levels, a decrease in chromatin higher-order packing states, an increased frequency of extended chromatin fiber formation, and deeper Con-A-responsive chromatin areas were observed in the hepatocytes of the NOD mice expressing insulin-dependent diabetes. Reduced amounts of total RNA were also found in the livers of these mice. Our findings for NOD mice expressing insulin-dependent diabetes are consistent with previously reported data for old-aged mice of the inbred strain A/Uni and may reflect changes in transcriptional activities associated with the stressful physiological demands on the liver during the expression of diabetes.

  7. Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes.

    PubMed

    Kasinsky, H E; Ellis, S; Martens, G; Ausió, J

    2014-12-01

    During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid-liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.

  8. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast.

    PubMed

    Steglich, Babett; Strålfors, Annelie; Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-03-01

    In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.

  9. The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

    PubMed Central

    Sellou, Hafida; Lebeaupin, Théo; Chapuis, Catherine; Smith, Rebecca; Hegele, Anna; Singh, Hari R.; Kozlowski, Marek; Bultmann, Sebastian; Ladurner, Andreas G.; Timinszky, Gyula; Huet, Sébastien

    2016-01-01

    Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo. PMID:27733626

  10. Interphase Chromosome Conformation and Chromatin-chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

    On a multi-mega base pair scale of the DNA, the arrangement of chromatin is non-random. In M10 epithelial cells, both telomere regions tend to be located towards the exterior of the chromosome domain, whereas the rest p-arm of the chromatin region towards the interior. In contrast, most of the q-arm of the chromatin is found in the peripheral of the domain. In lymphocytes, the p-arm chromatin regions towards the interior in close proximity with each other, whereas two q-arm regions are nearness in space. It indicates that G0 lymphocytes may lack secondary 3D chromatin folding. There chromatin folding patterns are consistent with our previous finding of non-random distribution of intra-chromosomal exchanges. In simulated microgravity conditions, the chromosome conformation may be altered and new regions in close proximity, especially to region 2 are suggested.

  11. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  12. Efficient skew-angle cladding-pumped tunable narrow-linewidth Yb-doped fiber laser.

    PubMed

    Jelger, Pär; Laurell, Fredrik

    2007-12-15

    A skew-angle cladding-pumped tunable Yb-doped fiber laser is presented. The laser was tunable over more than 30 nm, from 1022 to 1055 nm, by employing a volume Bragg grating in a retroreflector configuration as one of the cavity delimiters. Output powers in excess of 4.3 W were recorded with a spectral bandwidth of 5 GHz and an M(2) value below 1.3 over the whole tuning range.

  13. BIOCHEMICAL ANALYSES OF TRANSCRIPTIONAL REGULATORY MECHANISMS IN A CHROMATIN CONTEXT

    PubMed Central

    KONESKY, KASEY L.; LAYBOURN, PAUL J.

    2007-01-01

    We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how the degree of chromatin assembly in the absence and presence of histone H1 is measured using topological analysis and the use of micrococcal nuclease digestion performed to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further we describe the use sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates). PMID:17309835

  14. Multiple modes of chromatin remodeling by Forkhead box proteins.

    PubMed

    Lalmansingh, Avin S; Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2012-07-01

    Forkhead box (FOX) proteins represent a large family of transcriptional regulators unified by their DNA binding domain (DBD) known as a 'forkhead' or 'winged helix' domain. Over 40 FOX genes have been identified in the mammalian genome. FOX proteins share significant sequence similarities in the DBD which allow them to bind to a consensus DNA response element. However, their modes of action are quite diverse as they regulate gene expression by acting as pioneer factors, transcription factors, or both. This review focuses on the mechanisms of chromatin remodeling with an emphasis on three sub-classes-FOXA, FOXO, and FOXP members. FOXA proteins serve as pioneer factors to open up local chromatin structure and thereby increase accessibility of chromatin to factors regulating transcription. FOXP proteins, in contrast, function as classic transcription factors to recruit a variety of chromatin modifying enzymes to regulate gene expression. FOXO proteins represent a hybrid subclass having dual roles as pioneering factors and transcription factors. A subset of FOX proteins interacts with condensed mitotic chromatin and may function as 'bookmarking' agents to maintain transcriptional competence at specific genomic sites. The overall diversity in chromatin remodeling function by FOX proteins is related to unique structural motifs present within the DBD flanking regions that govern selective interactions with core histones and/or chromatin coregulatory proteins. This article is part of a Special Issue entitled: Chromatin in time and space.

  15. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin*

    PubMed Central

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Fedisch, Andreas; Schilcher, Pierre; Schmidt, Andreas; Imhof, Axel

    2016-01-01

    The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner. PMID:26811354

  16. A Broad Set of Chromatin Factors Influences Splicing

    PubMed Central

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  17. Chromatin dynamics: Interplay between remodeling enzymes and histone modifications

    PubMed Central

    Swygert, Sarah G.; Peterson, Craig L.

    2014-01-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. PMID:24583555

  18. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    PubMed

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  19. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    PubMed

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

  20. Fiber biology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton fiber cells arising from seed epidermis is the most important agricultural textile commodity in the world. To produce fully mature fibers, approximately two months of fiber developmental process are required. The timing of four distinctive fiber development stages consisting of initiation, ...

  1. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition

    PubMed Central

    1996-01-01

    We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture. PMID:8991083

  2. Transcription upregulation via force-induced direct stretching of chromatin

    NASA Astrophysics Data System (ADS)

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-12-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

  3. ATP dependent chromatin remodeling enzymes in embryonic stem cells.

    PubMed

    Saladi, Srinivas Vinod; de la Serna, Ivana L

    2010-03-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure.

  4. Chromatin dynamics during herpes simplex virus-1 lytic infection.

    PubMed

    Placek, Brandon J; Berger, Shelley L

    2010-01-01

    Herpes simplex virus type 1 is a DNA virus that can establish lytic infections in epithelial cells and latent infections in sensory neurons. Upon entry into the nucleus the genome of HSV-1 rapidly associates with histone proteins. Similar to the genomes of the cellular host, HSV-1 is subject to chromatin-based regulation of transcription and replication. However, unlike the host genome, nucleosomes appear to be underrepresented on the HSV genome. During lytic infection, when the genome is transcribed, the HSV-1 chromatin structure appears to be disorganized, and characterized by histone variant sub-types and post-translational modifications representative of active chromatin. In contrast, during latency, when the majority of the viral genome is transcriptionally silent, the chromatin is compacted into a regularly repeating, compact heterochromatic structure. Here we discuss recent studies that underscore the importance of chromatin regulation during the lytic phase of the HSV-1 life-cycle.

  5. HAMLET interacts with histones and chromatin in tumor cell nuclei.

    PubMed

    Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

    2003-10-24

    HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

  6. Diverse lamin-dependent mechanisms interact to control chromatin dynamics

    PubMed Central

    Camozzi, Daria; Capanni, Cristina; Cenni, Vittoria; Mattioli, Elisabetta; Columbaro, Marta; Squarzoni, Stefano; Lattanzi, Giovanna

    2014-01-01

    Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals.   Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation. PMID:25482195

  7. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  8. Chromatin insulators: lessons from the fly

    PubMed Central

    Gurudatta, B. V.

    2009-01-01

    Chromatin insulators are DNA–protein complexes with broad functions in nuclear biology. Drosophila has at least five different types of insulators; recent results suggest that these different insulators share some components that may allow them to function through common mechanisms. Data from genome-wide localization studies of insulator proteins indicate a possible functional specialization, with different insulators playing distinct roles in nuclear biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Current results suggest that insulators set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation and development. PMID:19752045

  9. Chromatin dynamics in kidney development and function.

    PubMed

    Bechtel-Walz, Wibke; Huber, Tobias B

    2014-06-01

    Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little is known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. A better understanding of the renal cell-specific epigenetic modifications and the interaction of different cell types to form this highly complex organ will not only help to better understand developmental defects and early loss of kidney function in children, but also help to understand and improve chronic disease progression, cell regeneration and renal aging.

  10. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

    This dissertation discuss two recent efforts, via a unique combination of structural bioinformatics and density functional theory, to unravel some of the details concerning how molecular machinery within the eukaryotic cell nucleus controls chromatin architecture. The first, a study of the 5-methylation of cytosine in 5'-CG-3' : 5'-CG-3' base-pair steps, reveals that the methyl groups roughen the local elastic energy landscape of the DNA. This enhances the probability of the canonical B-DNA structure transitioning into the undertwisted A-like and overtwisted C-like forms seen in nucleosomes, or looped segments of DNA bound to histones. The second part focuses on the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. The arginine residues are ob- served to apply a tunable mechanical load to the backbone, enabling precision-controlled activation of DNA deformations.

  11. Chromatin remodeling: from transcription to cancer.

    PubMed

    Yaniv, Moshe

    2014-09-01

    In this short review article, I have tried to trace the path that led my laboratory from the early studies of the structure of papova minichromosomes and transcription control to the investigation of chromatin remodeling complexes of the SWI/SNF family. I discuss briefly the genetic and biochemical studies that lead to the discovery of the SWI/SNF complex in yeast and drosophila and summarize some of the studies on the developmental role of the murine complex. The discovery of the tumor suppressor function of the SNF5/INI1/SMARCB1 gene in humans and the identification of frequent mutations in other subunits of this complex in different human tumors opened a fascinating field of research on this epigenetic regulator. The hope is to better understand tumor development and to develop novel treatments.

  12. Chromatin changes predict recurrence after radical prostatectomy

    PubMed Central

    Hveem, Tarjei S; Kleppe, Andreas; Vlatkovic, Ljiljana; Ersvær, Elin; Wæhre, Håkon; Nielsen, Birgitte; Kjær, Marte Avranden; Pradhan, Manohar; Syvertsen, Rolf Anders; Nesheim, John Arne; Liestøl, Knut; Albregtsen, Fritz; Danielsen, Håvard E

    2016-01-01

    Background: Pathological evaluations give the best prognostic markers for prostate cancer patients after radical prostatectomy, but the observer variance is substantial. These risk assessments should be supported and supplemented by objective methods for identifying patients at increased risk of recurrence. Markers of epigenetic aberrations have shown promising results in several cancer types and can be assessed by automatic analysis of chromatin organisation in tumour cell nuclei. Methods: A consecutive series of 317 prostate cancer patients treated with radical prostatectomy at a national hospital between 1987 and 2005 were followed for a median of 10 years (interquartile range, 7–14). On average three tumour block samples from each patient were included to account for tumour heterogeneity. We developed a novel marker, termed Nucleotyping, based on automatic assessment of disordered chromatin organisation, and validated its ability to predict recurrence after radical prostatectomy. Results: Nucleotyping predicted recurrence with a hazard ratio (HR) of 3.3 (95% confidence interval (CI), 2.1–5.1). With adjustment for clinical and pathological characteristics, the HR was 2.5 (95% CI, 1.5–4.1). An updated stratification into three risk groups significantly improved the concordance with patient outcome compared with a state-of-the-art risk-stratification tool (P<0.001). The prognostic impact was most evident for the patients who were high-risk by clinical and pathological characteristics and for patients with Gleason score 7. Conclusion: A novel assessment of epigenetic aberrations was capable of improving risk stratification after radical prostatectomy. PMID:27124335

  13. Baseline Chromatin Modification Levels May Predict ...

    EPA Pesticide Factsheets

    Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (03)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan­acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and5-hydroxymethylcytosine (5-hmC)-and the variability in the 03-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in 03-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic see

  14. Defining the multivalent functions of CTCF from chromatin state and three-dimensional chromatin interactions.

    PubMed

    Lu, Yiming; Shan, Guangyu; Xue, Jiguo; Chen, Changsheng; Zhang, Chenggang

    2016-07-27

    CCCTC-binding factor (CTCF) is a multi-functional protein that is assigned various, even contradictory roles in the genome. High-throughput sequencing-based technologies such as ChIP-seq and Hi-C provided us the opportunity to assess the multivalent functions of CTCF in the human genome. The location of CTCF-binding sites with respect to genomic features provides insights into the possible roles of this protein. Here we present the first genome-wide survey and characterization of three important functions of CTCF: enhancer insulator, chromatin barrier and enhancer linker. We developed a novel computational framework to discover the multivalent functions of CTCF based on chromatin state and three-dimensional chromatin architecture. We applied our method to five human cell lines and identified ∼46 000 non-redundant CTCF sites related to the three functions. Disparate effects of these functions on gene expression were found and distinct genomic features of these CTCF sites were characterized in GM12878 cells. Finally, we investigated the cell-type specificities of CTCF sites related to these functions across five cell types. Our study provides new insights into the multivalent functions of CTCF in the human genome.

  15. Note: Broadly tunable all-fiber ytterbium laser with 0.05 nm spectral width based on multimode interference filter

    SciTech Connect

    Mukhopadhyay, Pranab K. Gupta, Pradeep K.; Singh, Amarjeet; Sharma, Sunil K.; Bindra, Kushvinder S.; Oak, Shrikant M.

    2014-05-15

    A multimode interference filter with narrow transmission bandwidth and large self-imaging wavelength interval is constructed and implemented in an ytterbium doped fiber laser in all-fiber format for broad wavelength tunability as well as narrow spectral width of the output beam. The peak transmission wavelength of the multimode interference filter was tuned with the help of a standard in-fiber polarization controller. With this simple mechanism more than 30 nm (1038 nm–1070 nm) tuning range is demonstrated. The spectral width of the output beam from the laser was measured to be 0.05 nm.

  16. Persistent Chromatin Modifications Induced by High Fat Diet.

    PubMed

    Leung, Amy; Trac, Candi; Du, Juan; Natarajan, Rama; Schones, Dustin E

    2016-05-13

    Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

  17. Defining the chromatin signature of inducible genes in T cells

    PubMed Central

    2009-01-01

    Background Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation. Results To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation. Conclusions These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation. PMID:19807913

  18. A synergistic DNA logic predicts genome-wide chromatin accessibility

    PubMed Central

    Hashimoto, Tatsunori; Sherwood, Richard I.; Kang, Daniel D.; Rajagopal, Nisha; Barkal, Amira A.; Zeng, Haoyang; Emons, Bart J.M.; Srinivasan, Sharanya; Jaakkola, Tommi; Gifford, David K.

    2016-01-01

    Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when trained with DNase-seq data for a cell type is capable of predicting expected read counts of genome-wide chromatin accessibility at every base from DNA sequence alone, with the highest accuracy at hypersensitive sites shared across cell types. We confirm that a SCM accurately predicts chromatin accessibility for thousands of synthetic DNA sequences using a novel CRISPR-based method of highly efficient site-specific DNA library integration. SCMs are directly interpretable and reveal that a logic based on local, nonspecific synergistic effects, largely among pioneer TFs, is sufficient to predict a large fraction of cellular chromatin accessibility in a wide variety of cell types. PMID:27456004

  19. Chromatinization of the KSHV Genome During the KSHV Life Cycle

    PubMed Central

    Uppal, Timsy; Jha, Hem C.; Verma, Subhash C.; Robertson, Erle S.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle. PMID:25594667

  20. Fractal Characterization of Chromatin Decompaction in Live Cells

    PubMed Central

    Yi, Ji; Stypula-Cyrus, Yolanda; Blaha, Catherine S.; Roy, Hemant K.; Backman, Vadim

    2015-01-01

    Chromatin organization has a fundamental impact on the whole spectrum of genomic functions. Quantitative characterization of the chromatin structure, particularly at submicron length scales where chromatin fractal globules are formed, is critical to understanding this structure-function relationship. Such analysis is currently challenging due to the diffraction-limited resolution of conventional light microscopy. We herein present an optical approach termed inverse spectroscopic optical coherence tomography to characterize the mass density fractality of chromatin, and we apply the technique to observe chromatin decompaction in live cells. The technique makes it possible for the first time, to our knowledge, to sense intracellular morphology with length-scale sensitivity from ∼30 to 450 nm, thus primarily probing the higher-order chromatin structure, without resolving the actual structures. We used chromatin decompaction due to inhibition of histone deacytelases and measured the subsequent changes in the fractal dimension of the intracellular structure. The results were confirmed by transmission electron microscopy and confocal fluorescence microscopy. PMID:26636933

  1. Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

    PubMed

    Chen, Yu-Fan; Chou, Chia-Ching; Gartenberg, Marc R

    2016-08-01

    Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

  2. Stress-induced structural changes in plant chromatin.

    PubMed

    Probst, Aline V; Mittelsten Scheid, Ortrun

    2015-10-01

    Stress defense in plants is elaborated at the level of protection and adaptation. Dynamic changes in sophisticated chromatin substructures and concomitant transcriptional changes play an important role in response to stress, as illustrated by the transient rearrangement of compact heterochromatin structures or the modulation of chromatin composition and modification upon stress exposure. To connect cytological, developmental, and molecular data around stress and chromatin is currently an interesting, multifaceted, and sometimes controversial field of research. This review highlights some of the most recent findings on nuclear reorganization, histone variants, histone chaperones, DNA- and histone modifications, and somatic and meiotic heritability in connection with stress.

  3. DNA packing in chromatine, a manifestation of the Bonnet transformation.

    PubMed

    Blum, Z; Lidin, S

    1988-08-01

    The packing of DNA is described using the formalism of differential geometry. Winding of the DNA double helix around the histone 2-5 octamer forming a nucleosome and the condensation of the so-formed bead-on-a-string chromatine aided by histone 1 is interpreted as two consecutive isometric, i.e. Bonnet, transformations. The DNA double helix can be approximated to a helicoid which can be transformed isometrically to a catenoid, an approximation of the nucleosome. Owing to the organization of the histone octamer the extended chromatine takes a helicoidal shape allowing a second Bonnet transformation to consummate the condensation into a chromatine fibre.

  4. Fiber optic temperature sensing with enhanced sensitivity based on spectral interferometry

    NASA Astrophysics Data System (ADS)

    Militky, J.; Kadulova, M.; Ciprian, D.; Hlubina, P.

    2017-01-01

    Temperature sensing with enhanced sensitivity based on the spectral interference of polarization modes in a highly birefringent (HB) fiber is proposed and demonstrated. A temperature sensor employs a tandem configuration of a birefringent quartz crystal and HB fiber placed between an analyzer and a polarizer. In the setup a modified channeled spectrum is generated, which shifts with the temperature change of the sensing part of the HB fiber. We analyze the measurement method theoretically and show that the sensitivity of the temperature sensing based on the wavelength interrogation is enhanced in comparison to a standard method with a fiber interferometer. We also demonstrate the enhancement of the temperature sensitivity for three HB fibers under test. Experimental results show that the temperature sensing can reach a sensitivity of -0.30 nm/K, which is enhanced in comparison to -0.10 nm/K reached for a standard measurement.

  5. A Genetic Screen and Transcript Profiling Reveal a Shared Regulatory Program for Drosophila Linker Histone H1 and Chromatin Remodeler CHD1

    PubMed Central

    Kavi, Harsh; Lu, Xingwu; Xu, Na; Bartholdy, Boris A.; Vershilova, Elena; Skoultchi, Arthur I.; Fyodorov, Dmitry V.

    2015-01-01

    Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression. PMID:25628309

  6. Fiber Techniques

    ERIC Educational Resources Information Center

    Nalle, Leona

    1976-01-01

    Describes a course in fiber techniques, which covers design methods involving fibers and fabric, that students in the Art Department at Sleeping Giant Junior High School had the opportunity to learn. (Author/RK)

  7. Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages

    PubMed Central

    Wooden, Jessica; Ciborowski, Pawel

    2014-01-01

    The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a “one size fits all” rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM)a, we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10 minutes. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge. PMID:25220915

  8. Dietary Fiber

    MedlinePlus

    Fiber is a substance in plants. Dietary fiber is the kind you eat. It's a type of carbohydrate. You may also see it listed on a food label as soluble ... types have important health benefits. Good sources of dietary fiber include Whole grains Nuts and seeds Fruit and ...

  9. Ancestral Chromatin Configuration Constrains Chromatin Evolution on Differentiating Sex Chromosomes in Drosophila.

    PubMed

    Zhou, Qi; Bachtrog, Doris

    2015-06-01

    Sex chromosomes evolve distinctive types of chromatin from a pair of ancestral autosomes that are usually euchromatic. In Drosophila, the dosage-compensated X becomes enriched for hyperactive chromatin in males (mediated by H4K16ac), while the Y chromosome acquires silencing heterochromatin (enriched for H3K9me2/3). Drosophila autosomes are typically mostly euchromatic but the small dot chromosome has evolved a heterochromatin-like milieu (enriched for H3K9me2/3) that permits the normal expression of dot-linked genes, but which is different from typical pericentric heterochromatin. In Drosophila busckii, the dot chromosomes have fused to the ancestral sex chromosomes, creating a pair of 'neo-sex' chromosomes. Here we collect genomic, transcriptomic and epigenomic data from D. busckii, to investigate the evolutionary trajectory of sex chromosomes from a largely heterochromatic ancestor. We show that the neo-sex chromosomes formed <1 million years ago, but nearly 60% of neo-Y linked genes have already become non-functional. Expression levels are generally lower for the neo-Y alleles relative to their neo-X homologs, and the silencing heterochromatin mark H3K9me2, but not H3K9me3, is significantly enriched on silenced neo-Y genes. Despite rampant neo-Y degeneration, we find that the neo-X is deficient for the canonical histone modification mark of dosage compensation (H4K16ac), relative to autosomes or the compensated ancestral X chromosome, possibly reflecting constraints imposed on evolving hyperactive chromatin in an originally heterochromatic environment. Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration.

  10. Replication forks, chromatin loops and dormant replication origins.

    PubMed

    Blow, J Julian; Ge, Xin Quan

    2008-01-01

    When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.

  11. Chromatin remodeling in DNA double-strand break repair.

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

    ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.

  12. Histone variants and chromatin assembly in plant abiotic stress responses.

    PubMed

    Zhu, Yan; Dong, Aiwu; Shen, Wen-Hui

    2013-01-01

    Genome organization into nucleosomes and higher-order chromatin structures has profound implications for the regulation of gene expression, DNA replication and repair. The structure of chromatin can be remodeled by several mechanisms; among others, nucleosome assembly/disassembly and replacement of canonical histones with histone variants constitute important ones. In this review, we provide a brief description on the current knowledge about histone chaperones involved in nucleosome assembly/disassembly and histone variants in Arabidopsis thaliana. We discuss recent advances in revealing crucial functions of histone chaperones, nucleosome assembly/disassembly and histone variants in plant response to abiotic stresses. It appears that chromatin structure remodeling may provide a flexible, global and stable means for the regulation of gene transcription to help plants more effectively cope with environmental stresses. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  13. A multiplexed system for quantitative comparisons of chromatin landscapes

    PubMed Central

    van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

    2015-01-01

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

  14. A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

    PubMed

    van Galen, Peter; Viny, Aaron D; Ram, Oren; Ryan, Russell J H; Cotton, Matthew J; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Carroll, Kaitlin M; Cross, Michael B; Levine, Ross L; Bernstein, Bradley E

    2016-01-07

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

  15. Regulation of meiotic chromatin loop size by chromosomal position.

    PubMed Central

    Heng, H H; Chamberlain, J W; Shi, X M; Spyropoulos, B; Tsui, L C; Moens, P B

    1996-01-01

    At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8610120

  16. Nuclear morphometry and chromatin textural characteristics of basal cell carcinoma.

    PubMed

    Mendaçolli, Paola Jung; Brianezi, Gabrielli; Schmitt, Juliano Vilaverde; Marques, Mariângela Esther Alencar; Miot, Hélio Amante

    2015-01-01

    Histological subtypes of basal cell carcinoma have biological, evolutionary and distinct prognostic behavior. The analysis of characteristics of the nucleus can provide data on their cellular physiology and behavior. The authors of this study evaluated nuclear morphological parameters and textural patterns of chromatin from different subtypes of basal cell carcinoma: nodular (n=37), superficial (n=28) and sclerodermiform (n=28). The parameters were compared between neoplasms' subtypes and with unaffected adjacent basal epithelium. Nuclear area and diameter of sclerodermiform neoplasms were superior to the other subtypes. Chromatin's color intensity and fractal dimension were less intense in superficial subtypes. Nuclear roundness and chromatin's entropy presented lower values in tumors than in normal epithelium. There was significant correlation between morphological and textural variables of normal skin and tumors. Morphometric elements and textural chromatin's homogeneity of basal cell carcinomas may be related to evolutionary, biological and behavior particularities related to each histotype.

  17. Roles and activities of chromatin remodeling ATPases in plants.

    PubMed

    Han, Soon-Ki; Wu, Miin-Feng; Cui, Sujuan; Wagner, Doris

    2015-07-01

    Chromatin remodeling ATPases and their associated complexes can alter the accessibility of the genome in the context of chromatin by using energy derived from the hydrolysis of ATP to change the positioning, occupancy and composition of nucleosomes. In animals and plants, these remodelers have been implicated in diverse processes ranging from stem cell maintenance and differentiation to developmental phase transitions and stress responses. Detailed investigation of their roles in individual processes has suggested a higher level of selectivity of chromatin remodeling ATPase activity than previously anticipated, and diverse mechanisms have been uncovered that can contribute to the selectivity. This review summarizes recent advances in understanding the roles and activities of chromatin remodeling ATPases in plants.

  18. HACking the centromere chromatin code: insights from human artificial chromosomes.

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations.

  19. HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype

    PubMed Central

    Reeves, Raymond

    2009-01-01

    I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed. PMID:19748605

  20. Chromatin Insulators: Linking genome organization to cellular function

    PubMed Central

    Phillips-Cremins, Jennifer E.; Corces, Victor G.

    2013-01-01

    A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome. PMID:23706817

  1. Surface plasmon resonance sensor based on grapefruit-type photonic crystal fiber with silver nano-film

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Zheng, Yibo; Wang, Yuan; Yao, Jianquan

    2016-11-01

    In this letter, surface plasmon resonance sensors based on grapefruit-type photonic crystal fiber with different silver nano-film structure have been analyzed and compared though the finite element method (FEM). Numerical results show that excellent sensor resolution of 7.143×10-5RIU can be achieved as the thickness of the filling silver film is 30nm by spectrum detection method.

  2. Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster.

    PubMed

    Chereji, Răzvan V; Kan, Tsung-Wai; Grudniewska, Magda K; Romashchenko, Alexander V; Berezikov, Eugene; Zhimulev, Igor F; Guryev, Victor; Morozov, Alexandre V; Moshkin, Yuri M

    2016-02-18

    Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to 'phasing' off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.

  3. Irregular orientation of nucleosomes in the well-defined chromatin plates of metaphase chromosomes.

    PubMed

    Castro-Hartmann, Pablo; Milla, Maria; Daban, Joan-Ramon

    2010-05-18

    In previous studies with partially denatured metaphase chromosomes, we detected platelike structures instead of the chromatin fibers currently considered in different structural models for chromosomes. Here we have observed that dilution of compact metaphase chromosomes with hyposmotic solutions can transform whole chromatids into extended plates formed by many layers. Since this treatment is soft and it does not change the ionic conditions, these observations indicate that native chromosomes are formed by stacked plates. This strengthens our hypothesis about the multilayer structure of chromosomes, which was originally based on results obtained using stronger denaturing conditions. We have investigated the structure of plates emanated from chromosomes using electron tomography. Our three-dimensional reconstructions demonstrate conclusively that the surface of the plates is very smooth and do not show repetitive structures supporting any regular organization of nucleosomes; even the nucleosomes in plate edges show irregular orientations. Furthermore, we have used polarizing microscopy for the study of whole chromosomes in metaphase cells in aqueous solution. Our results show that condensed chromosomes are not birefringent under structuring ionic conditions similar to those used with plates. This observation is incompatible with the existence of parallel columns of nucleosomes within chromosomes. In summary, we have not detected any regular orientation of nucleosomes, but at the same time, our results indicate that the bulk of chromatin in native chromosomes is organized forming very well-defined plates, in which the nucleosomes of the successive layers are interdigitated. Presumably, this dense structure is required for safe transfer of DNA to daughter cells.

  4. Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

    PubMed

    Brackley, Chris A; Liebchen, Benno; Michieletto, Davide; Mouvet, Francois; Cook, Peter R; Marenduzzo, Davide

    2017-03-28

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

  5. Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.

    PubMed

    Bošković, Ana; Eid, André; Pontabry, Julien; Ishiuchi, Takashi; Spiegelhalter, Coralie; Raghu Ram, Edupuganti V S; Meshorer, Eran; Torres-Padilla, Maria-Elena

    2014-05-15

    The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.

  6. A map of open chromatin in human pancreatic islets

    PubMed Central

    Gaulton, Kyle J.; Nammo, Takao; Pasquali, Lorenzo; Simon, Jeremy M.; Giresi, Paul G.; Fogarty, Marie P.; Panhuis, Tami M.; Mieczkowski, Piotr; Secchi, Antonio; Bosco, Domenico; Berney, Thierry; Montanya, Eduard; Mohlke, Karen L.; Lieb, Jason D.; Ferrer, Jorge

    2010-01-01

    Tissue-specific transcriptional regulation is central to human disease1. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)2–4 coupled with high-throughput sequencing. We identified ~80,000 open chromatin sites. Comparison of islet FAIRE-seq to five non-islet cell lines revealed ~3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes exhibiting islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes (T2D)5, is located in islet-selective open chromatin. We show that rs7903146 heterozygotes exhibit allelic imbalance in islet FAIRE signal, and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements, and show that FAIRE-seq can guide identification of regulatory variants important for disease. PMID:20118932

  7. Isolation and Proteomics Analysis of Barley Centromeric Chromatin Using PICh.

    PubMed

    Zeng, Zixian; Jiang, Jiming

    2016-06-03

    Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum vulgare) by using a 2'-deoxy-2'fluoro-ribonucleotides (2'-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barley centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alpha-cenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed.

  8. Fragmentation of chromatin with 125I radioactive disintegrations.

    PubMed Central

    Turner, G N; Nobis, P; Dewey, W C

    1976-01-01

    The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated. PMID:963201

  9. A WD-Repeat Protein Stabilizes ORC Binding to Chromatin

    PubMed Central

    Shen, Zhen; Sathyan, Kizhakke M.; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

    2015-01-01

    SUMMARY Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  10. On the physical and chemical dynamics of chromatin

    NASA Astrophysics Data System (ADS)

    Apratim, Manjul

    The research performed leading to this dissertation is an endeavor to explore two broad classes of developmental phenomena in the chromatin complex in eukaryotic cells---physical, for instance, long range interactions between enhancers and promoters, and chemical, such as epigenetic chromatin silencing. I begin by introducing the reader to both types of phenomena, and then set the stage for our strategy in the exploration of the physical side of these processes by creating a new machinery from existing pieces of polymer physics. I then make a brief foray into theoretical realms in an attempt to answer the question of what kinds of conformations of polymers dominate in what regimes. Subsequently, I proceed to consider the problem of analyzing and interpreting data from a major technique of probing the behavior of the chromatin complex in vivo --- Chromosome Conformation Capture --- towards which end we have developed and implemented a new and robust algorithm called 'G.R.O.M.A.T.I.N.'. Subsequently, I explore how similar ideas may be invoked in the analysis of direct microscopic observations of native chromatin structure via Fluorescence in situ Hybridization. Following this, I look at the problems of epigenetic chromatin silencing domain formation and stability in the presence of titration feedback and of stochastic noise, and demonstrate how the widely accepted polymerization model of silencing is consistent with Chromatin Immunoprecipitation data from silencing domains in budding yeast. I finally conclude with musings on recent evidence pinpointing the need to unify the physical and chemical pictures into one grand formulation.

  11. Alteration of Large-Scale Chromatin Structure by Estrogen Receptor

    PubMed Central

    Nye, Anne C.; Rajendran, Ramji R.; Stenoien, David L.; Mancini, Michael A.; Katzenellenbogen, Benita S.; Belmont, Andrew S.

    2002-01-01

    The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily important in human physiology and disease, recruits coactivators which modify local chromatin structure. Here we describe effects of ER on large-scale chromatin structure as visualized in live cells. We targeted ER to gene-amplified chromosome arms containing large numbers of lac operator sites either directly, through a lac repressor-ER fusion protein (lac rep-ER), or indirectly, by fusing lac repressor with the ER interaction domain of the coactivator steroid receptor coactivator 1. Significant decondensation of large-scale chromatin structure, comparable to that produced by the ∼150-fold-stronger viral protein 16 (VP16) transcriptional activator, was produced by ER in the absence of estradiol using both approaches. Addition of estradiol induced a partial reversal of this unfolding by green fluorescent protein-lac rep-ER but not by wild-type ER recruited by a lac repressor-SRC570-780 fusion protein. The chromatin decondensation activity did not require transcriptional activation by ER nor did it require ligand-induced coactivator interactions, and unfolding did not correlate with histone hyperacetylation. Ligand-induced coactivator interactions with helix 12 of ER were necessary for the partial refolding of chromatin in response to estradiol using the lac rep-ER tethering system. This work demonstrates that when tethered or recruited to DNA, ER possesses a novel large-scale chromatin unfolding activity. PMID:11971975

  12. Brain neuronal chromatin responses in acute soman intoxicated rats.

    PubMed

    Martin, L J; Doebler, J A; Wall, T J; Shih, T M; Anthony, A

    1986-08-01

    Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50 = 135 micrograms/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.

  13. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

    Ahanger, Sajad H.; Günther, Katharina; Weth, Oliver; Bartkuhn, Marek; Bhonde, Ramesh R.; Shouche, Yogesh S.; Renkawitz, Rainer

    2014-01-01

    Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

  14. Chromatin topology is coupled to Polycomb group protein subnuclear organization

    PubMed Central

    Wani, Ajazul H.; Boettiger, Alistair N.; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan I.; Zhuang, Xiaowei; Kingston, Robert E.; Francis, Nicole J.

    2016-01-01

    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions. PMID:26759081

  15. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

    Li, Wangzhi; Wu, Jie; Kim, Sang-Yong; Zhao, Ming; Hearn, Stephen A.; Zhang, Michael Q.; Meistrich, Marvin L.

    2014-01-01

    One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling. PMID:24818823

  16. Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

    The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development. Images PMID:7312627

  17. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.

  18. Micronucleus formation during chromatin condensation and under apoptotic conditions.

    PubMed

    Kiraly, Gabor; Simonyi, Athene S; Turani, Melinda; Juhasz, Istvan; Nagy, Gabor; Banfalvi, Gaspar

    2017-02-01

    In early S phase the newly replicated DNA is folded back to increasingly compact structures. The process of chromatin condensation inside the nucleus starts with the formation of a micronucleus observed in five established cell lines (K562, CHO, Indian muntjac, murine preB and SCC). Supercoiling of chromatin generates a polarized end-plate region extruded from the nucleus. The extruded chromatin is turned around itself forming the head portion (micronucleus) visible by fluorescence microscopy until the middle of S phase when chromatin structures are succeeded by distinguishable early forms of chromosomes. The generation of micronuclei upon apoptotic treatment was achieved by the methotrexate (MTX) treatment of cells. A close correlation was found between the frequency of micronucleus and MTX concentration, with low frequency at low (0.1 µM) and increasingly higher frequency between 1 and 100 µM concentrations. Characteristic deformation and shrinkage of nuclei indicated apoptosis. High MTX concentration (100 µM) caused the enlargement and necrotic disruption of nuclei. Inhibition of DNA synthesis during replicative DNA synthesis by biotinylated nucleotide prevented the formation of metaphase chromosomes and elevated the frequency of early intermediates of chromosome condensation including micronucleus formation. Based on these observations the micronucleus is regarded as: (a) a regularly occuring element of early chromatin condensation and (b) a typical sign of nuclear membrane damage under toxic conditions. Explanation is given why the micronucleus is hidden in nuclei under normal chromatin condensation and why chromatin motifs including micronuclei become visible upon cellular damage.

  19. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.

    PubMed

    Jeffery, Daniel C B; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.

  20. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    PubMed Central

    Jeffery, Daniel CB; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly. PMID:25602519

  1. Remodeling of chromatin under low intensity diffuse ultrasound.

    PubMed

    Noriega, Sandra; Budhiraja, Gaurav; Subramanian, Anuradha

    2012-08-01

    A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms.

  2. Chromatin remodeling and bivalent histone modifications in embryonic stem cells.

    PubMed

    Harikumar, Arigela; Meshorer, Eran

    2015-12-01

    Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.

  3. The Circadian NAD+ Metabolism: Impact on Chromatin Remodeling and Aging

    PubMed Central

    Bessho, Yasumasa

    2016-01-01

    Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+ is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+ metabolism, and aging/age-related diseases. In addition, the interventions of NAD+ metabolism for the prevention and treatment of aging and age-related diseases are also discussed. PMID:28050554

  4. Micron-scale coherence in interphase chromatin dynamics

    PubMed Central

    Zidovska, Alexandra; Weitz, David A.; Mitchison, Timothy J.

    2013-01-01

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4–5 µm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces. PMID:24019504

  5. The role of noncoding RNAs in chromatin regulation during differentiation.

    PubMed

    Nahkuri, Satu; Paro, Renato

    2012-01-01

    A myriad of nuclear noncoding RNAs (ncRNAs) have been discovered since the paradigm of RNAs as plain conveyors of protein translation was discarded. There is increasing evidence that at vital intersections of developmental pathways, ncRNAs target the chromatin modulating machinery to its site of action. However, the mechanistic details of processes involved are still largely unclear, and well-characterized metazoan ncRNA species implicated in chromatin regulation during differentiation remain few. Nevertheless, four major categories are slowly emerging: cis-acting antisense ncRNAs that flag the neighboring genes for the propagation of chromatin marks; allele-specific ncRNAs that perform similar tasks, but target larger loci that typically vary in size from hundreds of thousands of base pairs to a whole chromosome; structural ncRNAs proposed to act as scaffolds that couple chromatin shaping complexes of distinct functionalities; and cofactor ncRNAs with a capacity to inhibit or activate essential components of the intertwined chromatin and transcription apparatuses.

  6. Chromatin is an ancient innovation conserved between Archaea and Eukarya

    PubMed Central

    Ammar, Ron; Torti, Dax; Tsui, Kyle; Gebbia, Marinella; Durbic, Tanja; Bader, Gary D; Giaever, Guri; Nislow, Corey

    2012-01-01

    The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient. DOI: http://dx.doi.org/10.7554/eLife.00078.001 PMID:23240084

  7. Topological constraints strongly affect chromatin reconstitution in silico

    PubMed Central

    Brackley, C.A.; Allan, J.; Keszenman-Pereyra, D.; Marenduzzo, D.

    2015-01-01

    The fundamental building block of chromatin, and of chromosomes, is the nucleosome, a composite material made up from DNA wrapped around a histone octamer. In this study we provide the first computer simulations of chromatin self-assembly, starting from DNA and histone proteins, and use these to understand the constraints which are imposed by the topology of DNA molecules on the creation of a polynucleosome chain. We take inspiration from the in vitro chromatin reconstitution protocols which are used in many experimental studies. Our simulations indicate that during self-assembly, nucleosomes can fall into a number of topological traps (or local folding defects), and this may eventually lead to the formation of disordered structures, characterised by nucleosome clustering. Remarkably though, by introducing the action of topological enzymes such as type I and II topoisomerase, most of these defects can be avoided and the result is an ordered 10-nm chromatin fibre. These findings provide new insight into the biophysics of chromatin formation, both in the context of reconstitution in vitro and in terms of the topological constraints which must be overcome during de novo nucleosome formation in vivo, e.g. following DNA replication or repair. PMID:25432958

  8. Transcription upregulation via force-induced direct stretching of chromatin

    PubMed Central

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-01-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green-fluorescent-protein (GFP) tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription. PMID:27548707

  9. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  10. [Resolution of spatial constraints during replication of peripheral chromatin].

    PubMed

    Zhironkina, O A; Kurchashova, S Yu; Bratseva, A L; Cherepanynets, V D; Strelkova, O S; Belmont, A S; Kireev, I I

    2014-01-01

    Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.

  11. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

    Zhang, Kai; Li, Nan; Ainsworth, Richard I.; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  12. Extension of chromatin accessibility by nuclear matrix attachment regions

    NASA Astrophysics Data System (ADS)

    Jenuwein, Thomas; Forrester, William C.; Fernández-Herrero, Luis A.; Laible, Götz; Dull, Maude; Grosschedl, Rudolf

    1997-01-01

    Transcription of the variable region of the rearranged immunoglobulin μ gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer1. As this μ gene enhancer can establish local areas of accessible chromatin2, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking μ enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The μ enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibilty correlates with extended demethylation of the geμ enhancer to generate an extended domain of accessible chromatin.

  13. Interplay between modifications of chromatin and meiotic recombination hotspots.

    PubMed

    Brachet, Elsa; Sommermeyer, Vérane; Borde, Valérie

    2012-02-01

    Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called 'hotspots'. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

  14. Modification of enhancer chromatin: what, how and why?

    PubMed Central

    Calo, Eliezer; Wysocka, Joanna

    2013-01-01

    Emergence of form and function during embryogenesis arises in large part through cell type- and cell state- specific variation in gene expression patterns, mediated by specialized cis-regulatory elements called enhancers. Recent large-scale epigenomic mapping revealed unexpected complexity and dynamics of enhancer utilization patterns, with 400,000 putative human enhancers annotated by the ENCODE project alone. These large-scale efforts were largely enabled through understanding that enhancers share certain stereotypical chromatin features. However, an important question still lingers: What is the functional significance of enhancer chromatin modification? Here we give an overview of enhancer-associated modifications of histones and DNA, and discuss enzymatic activities involved in their dynamic deposition and removal. We describe potential downstream effectors of these marks and propose models for exploring functions of chromatin modification in regulating enhancer activity during development. PMID:23473601

  15. Spatial organization of chromatin domains and compartments in single chromosomes.

    PubMed

    Wang, Siyuan; Su, Jun-Han; Beliveau, Brian J; Bintu, Bogdan; Moffitt, Jeffrey R; Wu, Chao-ting; Zhuang, Xiaowei

    2016-08-05

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation.

  16. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  17. Balancing chromatin remodeling and histone modifications in transcription

    PubMed Central

    Petty, Emily; Pillus, Lorraine

    2013-01-01

    Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive cross-talk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relationships between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the ISWI and CHD1 chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast. PMID:23870137

  18. PARP1 orchestrates epigenetic events setting up chromatin domains.

    PubMed

    Ciccarone, Fabio; Zampieri, Michele; Caiafa, Paola

    2017-03-01

    Epigenetic events include reversible modifications of DNA and histone tails driving chromatin organization and thus transcription. The epigenetic regulation is a highly integrated process underlying the plasticity of the genomic information both in the context of complex physiological and pathological processes. The global regulatory aspects of epigenetic events are largely unknown. PARylation and PARP1 are recently emerging as multi-level regulatory effectors that modulate the topology of chromatin by orchestrating very different processes. This review focuses in particular on the role of PARP1 in epigenetics, trying to build a comprehensive perspective of its involvement in the regulation of epigenetic modifications of histones and DNA, contextualizing it in the global organization of chromatin domains in the nucleus.

  19. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

    PubMed

    Hu, Jialei; Donahue, Greg; Dorsey, Jean; Govin, Jérôme; Yuan, Zuofei; Garcia, Benjamin A; Shah, Parisha P; Berger, Shelley L

    2015-12-01

    Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination.

  20. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  1. Identifying different types of chromatin using Giemsa staining.

    PubMed

    Stockert, Juan C; Blázquez-Castro, Alfonso; Horobin, Richard W

    2014-01-01

    Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The same occurs in the case of BT and BB chromatids.In addition to discussing the use of Giemsa stain as a suitable method to reveal specific features of chromosome structure, some molecular processes and models are also described to explain Giemsa staining mechanisms of chromatin.

  2. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility

    PubMed Central

    Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak H.; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter J.; Kingston, Robert E.; Tolstorukov, Michael Y.

    2016-01-01

    Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365

  3. Chromatin dynamics during interphase explored by single particle tracking

    PubMed Central

    Levi, Valeria; Gratton, Enrico

    2009-01-01

    Our view of the structure and function of the interphase nucleus has drastically changed in the last years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin -initially considered a randomly entangled polymer- has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques significantly evolved during the last years allowing the observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectories analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained by using this novel approach to study chromatin dynamics. PMID:18461483

  4. Chromatin dynamics during interphase explored by single-particle tracking.

    PubMed

    Levi, Valeria; Gratton, Enrico

    2008-01-01

    Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics.

  5. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    SciTech Connect

    Smith, P.J.

    1986-03-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

  6. Conformational change in the chromatin remodelling protein MENT.

    PubMed

    Ong, Poh Chee; Golding, Sarah J; Pearce, Mary C; Irving, James A; Grigoryev, Sergei A; Pike, Debbie; Langendorf, Christopher G; Bashtannyk-Puhalovich, Tanya A; Bottomley, Stephen P; Whisstock, James C; Pike, Robert N; McGowan, Sheena

    2009-01-01

    Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

  7. Mortality ratios and life expectancy in X chromatin positive males.

    PubMed Central

    Price, W H; Clayton, J F; Collyer, S; de Mey, R

    1985-01-01

    In a prospective study of 466 X chromatin positive males an increase in mortality of about 50% has been observed. The increase is associated with a loss of about five years in life span. There is no convincing evidence that the increase is concentrated at any particular age group but this possibility could not be excluded. No effect of mode of ascertainment could be demonstrated. From this study we conclude that it is likely that the mortality experienced by chromatin positive males in general is at least 115% of that experienced by normal men and could be more than 200%. PMID:4039353

  8. Chromatin remodelling: the industrial revolution of DNA around histones.

    PubMed

    Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R

    2006-06-01

    Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.

  9. Chromatin structure and pre-mRNA processing work together.

    PubMed

    Jimeno-González, Silvia; Reyes, José C

    2016-05-26

    Chromatin is the natural context for transcription elongation. However, the elongating RNA polymerase II (RNAPII) is forced to pause by the positioned nucleosomes present in gene bodies. Here, we briefly discuss the current results suggesting that those pauses could serve as a mechanism to coordinate transcription elongation with pre-mRNA processing. Further, histone post-translational modifications have been found to regulate the recruitment of factors involved in pre-mRNA processing. This view highlights the important regulatory role of the chromatin context in the whole process of the mature mRNA synthesis.

  10. Chromatin modification and epigenetic reprogramming in mammalian development.

    PubMed

    Li, En

    2002-09-01

    The developmental programme of embryogenesis is controlled by both genetic and epigenetic mechanisms. An emerging theme from recent studies is that the regulation of higher-order chromatin structures by DNA methylation and histone modification is crucial for genome reprogramming during early embryogenesis and gametogenesis, and for tissue-specific gene expression and global gene silencing. Disruptions to chromatin modification can lead to the dysregulation of developmental processes, such as X-chromosome inactivation and genomic imprinting, and to various diseases. Understanding the process of epigenetic reprogramming in development is important for studies of cloning and the clinical application of stem-cell therapy.

  11. SANS spectra of the fractal supernucleosomal chromatin structure models

    NASA Astrophysics Data System (ADS)

    Ilatovskiy, Andrey V.; Lebedev, Dmitry V.; Filatov, Michael V.; Petukhov, Michael G.; Isaev-Ivanov, Vladimir V.

    2012-03-01

    The eukaryotic genome consists of chromatin—a nucleoprotein complex with hierarchical architecture based on nucleosomes, the organization of higher-order chromatin structures still remains unknown. Available experimental data, including SANS spectra we had obtained for whole nuclei, suggested fractal nature of chromatin. Previously we had built random-walk supernucleosomal models (up to 106 nucleosomes) to interpret our SANS spectra. Here we report a new method to build fractal supernucleosomal structure of a given fractal dimension or two different dimensions. Agreement between calculated and experimental SANS spectra was significantly improved, especially for model with two fractal dimensions—3 and 2.

  12. Do chromatin changes around a nascent double strand DNA break spread spherically into linearly non-adjacent chromatin?

    PubMed

    Savic, Velibor

    2013-01-01

    In the last decade, a lot has been done in elucidating the sequence of events that occur at the nascent double strand DNA break. Nevertheless, the overall structure formed by the DNA damage response (DDR) factors around the break site, the repair focus, remains poorly understood. Although most of the data presented so far only address events that occur in chromatin in cis around the break, there are strong indications that in mammalian systems it may also occur in trans, analogous to the recent findings showing this if budding yeast. There have been attempts to address the issue but the final proof is still missing due to lack of a proper experimental system. If found to be true, the spatial distribution of DDR factors would have a major impact on the neighboring chromatin both in cis and in trans, significantly affecting local chromatin function; gene transcription and potentially other functions.

  13. Two Fiber Optical Fiber Thermometry

    NASA Technical Reports Server (NTRS)

    Jones, Mathew R.; Farmer, Jeffery T.; Breeding, Shawn P.

    2000-01-01

    An optical fiber thermometer consists of an optical fiber whose sensing tip is given a metallic coating. The sensing tip of the fiber is essentially an isothermal cavity, so the emission from this cavity will be approximately equal to the emission from a blackbody. Temperature readings are obtained by measuring the spectral radiative heat flux at the end of the fiber at two wavelengths. The ratio of these measurements and Planck's Law are used to infer the temperature at the sensing tip. Optical fiber thermometers have high accuracy, excellent long-term stability and are immune to electromagnetic interference. In addition, they can be operated for extended periods without requiring re-calibration. For these reasons. it is desirable to use optical fiber thermometers in environments such as the International Space Station. However, it has recently been shown that temperature readings are corrupted by emission from the fiber when extended portions of the probe are exposed to elevated temperatures. This paper will describe several ways in which the reading from a second fiber can be used to correct the corrupted temperature measurements. The accuracy and sensitivity to measurement uncertainty will be presented for each method.

  14. ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin

    PubMed Central

    Lawrimore, Josh; Aicher, Joseph K.; Hahn, Patrick; Fulp, Alyona; Kompa, Ben; Vicci, Leandra; Falvo, Michael; Taylor, Russell M.; Bloom, Kerry

    2016-01-01

    ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis. PMID:26538024

  15. Functional Interplay Between Histone H1 and HMG Proteins in Chromatin

    PubMed Central

    Postnikov, Yuri V.; Bustin, Michael

    2015-01-01

    The dynamic interaction of nucleosome binding proteins with their chromatin targets is an important element in regulating the structure and function of chromatin. Histone H1 variants and High Mobility Group (HMG) proteins are ubiquitously expressed in all vertebrate cells, bind dynamically to chromatin, and are known to affect chromatin condensation and the ability of regulatory factors to access their genomic binding sites. Here, we review the studies that focus on the interactions between H1 and HMGs and highlight the functional consequences of the interplay between these architectural chromatin binding proteins. H1 and HMG proteins are mobile molecules that bind to nucleosomes as members of a dynamic protein network. All HMGs compete with H1 for chromatin binding sites, in a dose dependent fashion, but each HMG family has specific effects on the interaction of H1 with chromatin. The interplay between H1 and HMGs affects chromatin organization and plays a role in epigenetic regulation. PMID:26455954

  16. Epigenetic regulation and chromatin remodeling in learning and memory

    PubMed Central

    Kim, Somi; Kaang, Bong-Kiun

    2017-01-01

    Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms. PMID:28082740

  17. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-08

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.

  18. Analysis of chromatin structure at meiotic DSB sites in yeasts.

    PubMed

    Hirota, Kouji; Fukuda, Tomoyuki; Yamada, Takatomi; Ohta, Kunihiro

    2009-01-01

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion ofchromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

  19. Generation of bivalent chromatin domains during cell fate decisions

    PubMed Central

    2011-01-01

    Background In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. Results Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. Conclusions While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different. PMID:21645363

  20. Increased sperm ubiquitination correlates with abnormal chromatin integrity.

    PubMed

    Hodjat, M; Akhondi, M A; Al-Hasani, S; Mobaraki, M; Sadeghi, M R

    2008-09-01

    Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

  1. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  2. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  3. Alterations in chromatin structure during early sea urchin embryogenesis.

    PubMed Central

    Savić, A; Richman, P; Williamson, P; Poccia, D

    1981-01-01

    Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition. Images PMID:6943576

  4. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  5. Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

    PubMed

    Mata-Garrido, Jorge; Casafont, Iñigo; Tapia, Olga; Berciano, Maria T; Lafarga, Miguel

    2016-04-22

    There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of

  6. Fireblocking Fibers

    NASA Technical Reports Server (NTRS)

    1986-01-01

    PBI was originally developed for space suits. In 1980, the need for an alternative to asbestos and stricter government anti-pollution standards led to commercialization of the fire blocking fiber. PBI is used for auto racing driver suits and aircraft seat covers. The fiber does not burn in air, is durable and easily maintained. It has been specified by a number of airliners and is manufactured by Hoechst-Celanese Corporation.

  7. Dietary fiber.

    PubMed

    Madar, Z; Thorne, R

    1987-01-01

    Studies done on dietary fiber (DF) over the past five years are presented in this Review. The involvement of dietary fiber in the control of plasma glucose and lipid levels is now established. Two dietary fiber sources (soybean and fenugreek) were studied in our laboratory and are discussed herein. These sources were found to be potentially beneficial in the reduction of plasma glucose in non-insulin dependent diabetes mellitus subjects. They are shown to be acceptable by human subjects and are easy to use either in a mixture of milk products and in cooking. The mechanism by which dietary fiber alters the nutrient absorption is also discussed. The effect of DF on gastric emptying, transit time, adsorption and glucose transport may contribute to reducing plasma glucose and lipid levels. DF was found to be effective in controlling blood glucose and lipid levels of pregnant diabetic women. Dietary fiber may also be potentially beneficial in the reduction of exogenous insulin requirements in these subjects. However, increased consumption of DF may cause adverse side effects; the binding capabilities of fiber may affect nutrient availability, particularly that of minerals and prolonged and high DF dosage supplementation must be regarded cautiously. This is particularly true when recommending such a diet for pregnant or lactating women, children or subjects with nutritional disorders. Physiological effects of DF appear to depend heavily on the source and composition of fiber. Using a combination of DF from a variety of sources may reduce the actual mass of fiber required to obtain the desired metabolic effects and will result in a more palatable diet. Previously observed problems, such as excess flatus, diarrhea and mineral malabsorption would also be minimized.

  8. Nanocomposite Fibers

    DTIC Science & Technology

    2003-01-01

    attempts to prepare carbon nanotube , CNT, containing fiber material. Modulus and tenacity tests on experimentally prepared nanosilica filled PET...individual entities of nanofibers, such as carbon nanotubes and SiC whiskers, silica and clay, into polymers with the goal of producing new forms of...if carbon nanotube (CNT) particle implanted fibers are used, one would expect a great increase in the electrical conductivity of the so-reinforced

  9. Regulation of Nucleosome Stacking and Chromatin Compaction by the Histone H4 N-Terminal Tail-H2A Acidic Patch Interaction.

    PubMed

    Chen, Qinming; Yang, Renliang; Korolev, Nikolay; Liu, Chuan Fa; Nordenskiöld, Lars

    2017-03-16

    Chromatin folding and dynamics are critically dependent on nucleosome-nucleosome interactions with important contributions from internucleosome binding of the histone H4 N-terminal tail K16-R23 domain to the surface of the H2A/H2B dimer. The H4 Lys16 plays a pivotal role in this regard. Using in vitro reconstituted 12-mer nucleosome arrays, we have investigated the mechanism of the H4 N-terminal tail in maintaining nucleosome-nucleosome stacking and mediating intra- and inter-array chromatin compaction, with emphasis on the role of K16 and the positive charge region, R17-R23. Analytical ultracentrifugation sedimentation velocity experiments and precipitation assays were employed to analyze effects on chromatin folding and self-association, respectively. Effects on chromatin folding caused by various mutations and modifications at position K16 in the H4 histone were studied. Additionally, using charge-quenching mutations, we characterized the importance of the interaction of the residues within the H4 positive charge region R17-R23 with the H2A acidic patch of the adjacent nucleosome. Furthermore, crosslinking experiments were conducted to establish the proximity of the basic tail region to the acidic patch. Our data indicate that the positive charge and length of the side chain of H4 K16 are important for its access to the adjacent nucleosome in the process of nucleosome-nucleosome stacking and array folding. The location and orientation of the H4 R17-R23 domain on the H2A/H2B dimer surface of the neighboring nucleosome core particle (NCP) in the compacted chromatin fiber were established. The dominance of electrostatic interactions in maintaining intra-array interaction was demonstrated.

  10. 1 μJ, sub-500 fs chirped pulse amplification in a Tm-doped fiber system.

    PubMed

    Sims, R Andrew; Kadwani, Pankaj; Shah, Alex Sincore Lawrence; Richardson, Martin

    2013-01-15

    We demonstrate a Tm-doped fiber laser system producing ~300 fs pulses with 1 μJ energy, corresponding to peak powers greater than 3 MW. Pulses of 150 fs with 30 nm spectral bandwidth and 3 nJ pulse energy are generated in a Raman-soliton self-frequency shift amplifier, then stretched to ~160 ps using a chirped Bragg grating. The 60 MHz oscillator repetition rate is reduced to 100 kHz using an electro-optic modulator. After a single-mode fiber preamplifier and a large-mode-area fiber power amplifier, pulses were compressed using a folded Treacy grating setup to below 500 fs with up to 1 μJ pulse energy. To the best of our knowledge, this is the highest energy yet demonstrated as well as the first demonstration of peak powers exceeding 1 MW from a Tm:fiber laser system.

  11. Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information.

    PubMed

    Corpet, Armelle; Almouzni, Geneviève

    2009-01-01

    Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.

  12. Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

    PubMed

    Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E

    2016-10-06

    Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

  13. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites

    NASA Astrophysics Data System (ADS)

    Wang, Yejun; Maharana, Shovamayee; Wang, Michelle D.; Shivashankar, G. V.

    2014-03-01

    Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy.

  14. Comparison of the effect of UV laser radiation and of a radiomimetic substance on chromatin

    NASA Astrophysics Data System (ADS)

    Radulescu, Irina; Radu, Liliana; Serbanescu, Ruxandra; Nelea, V. D.; Martin, C.; Mihailescu, Ion N.

    1998-07-01

    The damages of the complex of deoxyribonucleic acid (DNA) and proteins from chromatin, produced by the UV laser radiation and/or by treatment with a radiomimetic substance, bleomycin, were compared. The laser radiation and bleomycin effects on chromatin structure were determined by the static and dynamic fluorimetry of chromatin complexes with the DNA specific ligand-- proflavine and by the analysis of tryptophan chromatin intrinsic fluorescence. Time resolved spectroscopy is a sensitive technique which allows to determine the excited state lifetimes of chromatin--proflavine complexes. Also, the percentage contributions to the fluorescence of proflavine, bound and unbound to chromatin DNA, were evaluated. The damages produced by the UV laser radiation on chromatin are similar with those of radiomimetic substance action and consists in DNA and proteins destruction. The DNA damage degree has been determined. The obtained results may constitute some indications in the laser utilization in radiochimiotherapy.

  15. Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro.

    PubMed

    Prusov, A N; Smirnova, T A; Kolomijtseva, G Ya

    2013-02-01

    In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100-200 nm; N-1) and partly decondensed (diameter of fibrils ~30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4-7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin

  16. In vivo chromatin accessibility correlates with gene silencing in Drosophila.

    PubMed Central

    Boivin, A; Dura, J M

    1998-01-01

    Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure. PMID:9832530

  17. Chromatin Memory in the Development of Human Cancers

    PubMed Central

    Yao, Yixin; Des Marais, Thomas L; Costa, Max

    2014-01-01

    Cancer is a complex disease with acquired genomic and epigenomic alterations that affect cell proliferation, viability and invasiveness. Almost all the epigenetic mechanisms including cytosine methylation and hydroxymethylation, chromatin remodeling and non-coding RNAs have been found associate with carcinogenesis and cancer specific expression profile. Altered histone modification as an epigenetic hallmark is frequently found in tumors. Understanding the epigenetic alterations induced by carcinogens or infectious agents may help us understand early epigenetic changes prior to the development of cancer. In this review, we focus on chromatin remodeling and the associated histone modifiers in the development of cancer; the application of these modifiers as a cancer therapy target in different clinical trial phases is also discussed. PMID:25606572

  18. Chromatin Assembly in a Yeast Whole-Cell Extract

    NASA Astrophysics Data System (ADS)

    Schultz, Michael C.; Hockman, Darren J.; Harkness, Troy A. A.; Garinther, Wendy I.; Altheim, Brent A.

    1997-08-01

    A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

  19. Cellular and Chromatin Dynamics of Antibody-Secreting Plasma Cells

    PubMed Central

    Bortnick, Alexandra; Murre, Cornelis

    2015-01-01

    Plasma cells are terminally differentiated B cells responsible for maintaining protective serum antibody titers. Despite their clinical importance, our understanding of the linear genomic features and chromatin structure of plasma cells is incomplete. The plasma cell differentiation program can be triggered by different signals and in multiple, diverse peripheral B cell subsets. This heterogeneity raises questions about the gene regulatory circuits required for plasma cell specification. Recently, new regulators of plasma cell differentiation have been identified and the enhancer landscapes of naïve B cells have been described. Other studies have revealed that the bone marrow niche harbors heterogeneous plasma cell subsets. Still undefined are the minimal requirements to become a plasma cell and what molecular features make peripheral B cell subsets competent to become antibody-secreting plasma cells. New technologies promise to reveal underlying chromatin configurations that promote efficient antibody secretion. PMID:26488117

  20. JOINING THE DOTS: FROM CHROMATIN REMODELING TO NEURONAL PLASTICITY

    PubMed Central

    Zocchi, Loredana; Sassone-Corsi, Paolo

    2010-01-01

    SUMMARY In recent years spectacular advances in the field of epigenetics have taken place. Multiple lines of evidence that connect epigenetic regulation to brain functions have been accumulating. Neurons daily convert a variety of external stimuli into rapid or long-lasting changes in gene expression. Control is achieved through several post-translational modifications that occur both on DNA and chromatin. Specific modifications mediate many developmental processes and adult brain functions, such as synaptic plasticity and memory. In this review, we focus on critical chromatin remodeling events that mediate long-lasting neuronal responses. The challenging goal is to reach sufficient understanding of these epigenetic pathways in the brain so that they may be useful for future development of specific pharmacological strategies. PMID:20471240

  1. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

    We have made considerable progress in chromatin reconstitution with very lysine rich histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized in intrinsically bent DNA region flaking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interactions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear Magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  2. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

    Despite of setbacks in the lack of neutrons for the proposed We have made considerable progress in chromatin reconstitution with the VLR histone H1/H5 and in understanding the dynamics of nucleosomes. A ferromagnetic fluid was developed to align biological molecules for structural studies using small-angle-neutron-scattering. We have also identified and characterized an intrinsically bent DNA region flanking the RNA polymerase I binding site of the ribosomal RNA gene in Physarum Polycephalum. Finally projects in progress are in the areas of studying the interatctions of histone H4 amino-terminus peptide 1-23 and acetylated 1-23 peptide with DNA using thermal denaturation; study of GGAAT repeats found in human centromeres using high resolution Nuclear magnetic Resonance and nuclease sentivity assay; and the role of histones and other sperm specific proteins with sperm chromatin.

  3. Proteomics and the genetics of sperm chromatin condensation

    PubMed Central

    Oliva, Rafael; Castillo, Judit

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies. PMID:21042303

  4. Base excision repair in chromatin: Insights from reconstituted systems

    PubMed Central

    Balliano, Angela J.; Hayes, Jeffrey J.

    2015-01-01

    The process of base excision repair has been completely reconstituted in vitro and structural and biochemical properties of the component enzymes thoroughly studied on naked DNA templates. More recent work in this field aims to understand how BER operates on the natural substrate, chromatin [1, 2]. Toward this end, a number of researchers, including the Smerdon group, have focused attention to understand how individual enzymes and reconstituted BER operate on nucleosome substrates. While nucleosomes were once thought to completely restrict access of DNA-dependent factors, the surprising finding from these studies suggests that at least some BER components can utilize target DNA bound within nucleosomes as substrates for their enzymatic processes. This data correlates well with both structural studies of these enzymes and our developing understanding of nucleosome conformation and dynamics. While more needs to be learned, these studies highlight the utility of reconstituted BER and chromatin systems to inform our understanding of in vivo biological processes. PMID:26411876

  5. A quantitative telomeric chromatin isolation protocol identifies different telomeric states

    NASA Astrophysics Data System (ADS)

    Grolimund, Larissa; Aeby, Eric; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; Lingner, Joachim

    2013-11-01

    Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.

  6. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

    PubMed Central

    Leppkes, Moritz; Maueröder, Christian; Hirth, Sebastian; Nowecki, Stefanie; Günther, Claudia; Billmeier, Ulrike; Paulus, Susanne; Biermann, Mona; Munoz, Luis E.; Hoffmann, Markus; Wildner, Dane; Croxford, Andrew L.; Waisman, Ari; Mowen, Kerri; Jenne, Dieter E.; Krenn, Veit; Mayerle, Julia; Lerch, Markus M.; Schett, Georg; Wirtz, Stefan; Neurath, Markus F.; Herrmann, Martin; Becker, Christoph

    2016-01-01

    Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts. PMID:26964500

  7. piRNA clusters and open chromatin structure

    PubMed Central

    2014-01-01

    Transposable elements (TEs) are major structural components of eukaryotic genomes; however, mobilization of TEs generally has negative effects on the host genome. To counteract this threat, host cells have evolved genetic and epigenetic mechanisms that keep TEs silenced. One such mechanism involves the Piwi-piRNA complex, which represses TEs in animal gonads either by cleaving TE transcripts in the cytoplasm or by directing specific chromatin modifications at TE loci in the nucleus. Most Piwi-interacting RNAs (piRNAs) are derived from genomic piRNA clusters. There has been remarkable progress in our understanding of the mechanisms underlying piRNA biogenesis. However, little is known about how a specific locus in the genome is converted into a piRNA-producing site. In this review, we will discuss a possible link between chromatin boundaries and piRNA cluster formation. PMID:25126116

  8. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis.

    PubMed

    Leppkes, Moritz; Maueröder, Christian; Hirth, Sebastian; Nowecki, Stefanie; Günther, Claudia; Billmeier, Ulrike; Paulus, Susanne; Biermann, Mona; Munoz, Luis E; Hoffmann, Markus; Wildner, Dane; Croxford, Andrew L; Waisman, Ari; Mowen, Kerri; Jenne, Dieter E; Krenn, Veit; Mayerle, Julia; Lerch, Markus M; Schett, Georg; Wirtz, Stefan; Neurath, Markus F; Herrmann, Martin; Becker, Christoph

    2016-03-11

    Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts.

  9. Chromatin and Epigenetics at the Forefront: Finding Clues among Peaks

    PubMed Central

    Shi, Yang

    2016-01-01

    The Keystone Symposium on Chromatin and Epigenetics, organized by Luciano Di Croce (Center for Genomic Regulation, Spain) and Yang Shi (Harvard Medical School, USA), took place 20 to 24 March 2016 at Whistler (British Columbia, Canada). The symposium brought together some of the most outstanding scientists studying how chromatin structure and epigenetic mechanisms regulate gene function in both development and disease. Junior scientists had the opportunity to interact with experienced researchers by presenting their work and discussing ideas and novel hypotheses. In order to foster interaction and networking, the scientific agenda was balanced with an extended social agenda. This meeting review describes several of the most provocative and exciting talks from the symposium, revealing how fast this research field is evolving and the profound impact it will have on human health. PMID:27402863

  10. Characterization of brain cell nuclei with decondensed chromatin.

    PubMed

    Yu, Ping; McKinney, Elizabeth C; Kandasamy, Muthugapatti M; Albert, Alexandria L; Meagher, Richard B

    2015-07-01

    Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuN-Neg nuclei. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease.

  11. Light scattering measurements supporting helical structures for chromatin in solution.

    PubMed

    Campbell, A M; Cotter, R I; Pardon, J F

    1978-05-01

    Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.

  12. Chromatin condensation of Xist genomic loci during oogenesis in mice.

    PubMed

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2015-12-01

    Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.

  13. Photovoltaic fibers

    NASA Astrophysics Data System (ADS)

    Gaudiana, Russell; Eckert, Robert; Cardone, John; Ryan, James; Montello, Alan

    2006-08-01

    It was realized early in the history of Konarka that the ability to produce fibers that generate power from solar energy could be applied to a wide variety of applications where fabrics are utilized currently. These applications include personal items such as jackets, shirts and hats, to architectural uses such as awnings, tents, large covers for cars, trucks and even doomed stadiums, to indoor furnishings such as window blinds, shades and drapes. They may also be used as small fabric patches or fiber bundles for powering or recharging batteries in small sensors. Power generating fabrics for clothing is of particular interest to the military where they would be used in uniforms and body armor where portable power is vital to field operations. In strong sunlight these power generating fabrics could be used as a primary source of energy, or they can be used in either direct sunlight or low light conditions to recharge batteries. Early in 2002, Konarka performed a series of proof-of-concept experiments to demonstrate the feasibility of building a photovoltaic cell using dye-sensitized titania and electrolyte on a metal wire core. The approach taken was based on the sequential coating processes used in making fiber optics, namely, a fiber core, e.g., a metal wire serving as the primary electrode, is passed through a series of vertically aligned coating cups. Each of the cups contains a coating fluid that has a specific function in the photocell. A second wire, used as the counter electrode, is brought into the process prior to entering the final coating cup. The latter contains a photopolymerizable, transparent cladding which hardens when passed through a UV chamber. Upon exiting the UV chamber, the finished PV fiber is spooled. Two hundred of foot lengths of PV fiber have been made using this process. When the fiber is exposed to visible radiation, it generates electrical power. The best efficiency exhibited by these fibers is 6% with an average value in the 4

  14. Liquid seal for temperature sensing with fiber-optic refractometers.

    PubMed

    Xu, Ben; Li, Jianqing; Li, Yi; Xie, Jianglei; Dong, Xinyong

    2014-08-13

    Liquid sealing is an effective method to convert a fiber-optic refractometer into a simple and highly sensitive temperature sensor. A refractometer based on the thin-core fiber modal interferometer is sealed in a capillary tube filled with Cargille oil. Due to the thermo-optic effect of the sealing liquid, the high refractive-index sensitivity refractometer is subsequently sensitive to the ambient temperature. It is found that the liquid-sealed sensor produces a highest sensitivity of -2.30 nm/°C, which is over 250 times higher than its intrinsic sensitivity before sealing and significantly higher than that of a grating-based fiber sensors. The sensing mechanisms, including the incidental temperature-induced strain effect, are analyzed in detail both theoretically and experimentally. The liquid sealing technique is easy and low cost, and makes the sensor robust and insensitive to the surrounding refractive index. It can be applied to other fiber-optic refractometers for temperature sensing.

  15. Predicting chromatin architecture from models of polymer physics.

    PubMed

    Bianco, Simona; Chiariello, Andrea M; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2017-01-09

    We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the Sox9 locus, linked to human congenital disorders. The model in-silico predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.

  16. (Neutron scatter studies of chromatin structure related to function)

    SciTech Connect

    Bradbury, E.M.

    1990-01-01

    This study is concerned with the application of neutron scatter techniques to the different structural states of nucleosomes and chromatin with the long term objective of understanding how the enormous lengths of DNA are folded into chromosomes. Micrococcal nuclease digestion kinetics have defined two subnucleosome particles; the chromatosome with 168 bp DNA, the histone octamer and one H1 and the nucleosome core particle with 146 bp DNA and the histone octamer. As will be discussed, the structure of the 146 bp DNA core particle is known in solution at low resolution from neutron scatter studies and in crystals. Based on this structure, the authors have a working model for the chromatosome and the mode of binding of H1. In order to define the structure of the nucleosome and also the different orders of chromatin structures they need to know the paths of DNA that link nucleosomes and the factors associated with chromosome functions that act on those DNA paths. The major region for this situation is the inherent variabilities in nucleosome DNA sequences, in the histone subtypes and their states of chemical modification and in the precise locations of nucleosomes. Such variabilities obscure the underlying principles that govern the packaging of DNA into the different structural states of nucleosomes and chromatin. The only way to elucidate these principles is to study the structures of nucleosomes and oligonucleosomes that are fully defined. They have largely achieved these objectives.

  17. Terahertz Dynamics in Human Cells and Their Chromatin.

    PubMed

    Longo, M; Marconi, M; Orecchini, A; Petrillo, C; Monaco, G; Calvitti, M; Pirisinu, I; Romani, R; Sacchetti, F; Sebastiani, F; Zanatta, M; Paciaroni, A

    2014-07-03

    The terahertz dynamics of human cells of the U937 line and their chromatin has been investigated by high-resolution inelastic X-ray scattering. To highlight its dynamical features in situ, nuclear DNA has been stained by uranyl-acetate salt. The general behavior of the collective dynamics of the whole cell is quite similar to that of bulk water, with a nearly wavevector-independent branch located at about 5 meV and a propagating mode with a linear trend corresponding to a speed of sound of 2900 ± 100 m/s. We provide the first experimental evidence for the existence of two branches also in the dispersion curves of chromatin. The high-energy mode displays an acoustic-like behavior with a sound velocity similar to unstained cells, but in this case the branch likely originates from the superposition of intramolecular DNA optic modes. A low-energy optic-like branch, distinctive of the chromatin moiety, is found at about 2.5 meV.

  18. Chromatin signaling in muscle stem cells: interpreting the regenerative microenvironment

    PubMed Central

    Brancaccio, Arianna; Palacios, Daniela

    2015-01-01

    Muscle regeneration in the adult occurs in response to damage at expenses of a population of adult stem cells, the satellite cells. Upon injury, either physical or genetic, signals released within the satellite cell niche lead to the commitment, expansion and differentiation of the pool of muscle progenitors to repair damaged muscle. To achieve this goal satellite cells undergo a dramatic transcriptional reprogramming to coordinately activate and repress specific subset of genes. Although the epigenetics of muscle regeneration has been extensively discussed, less emphasis has been put on how extra-cellular cues are translated into the specific chromatin reorganization necessary for progression through the myogenic program. In this review we will focus on how satellite cells sense the regenerative microenvironment in physiological and pathological circumstances, paying particular attention to the mechanism through which the external stimuli are transduced to the nucleus to modulate chromatin structure and gene expression. We will discuss the pathways involved and how alterations in this chromatin signaling may contribute to satellite cells dysfunction during aging and disease. PMID:25904863

  19. Epigenetics and chromatin plasticity in embryonic stem cells

    PubMed Central

    Přikrylová, Terézia; Pacherník, Jiří; Kozubek, Stanislav; Bártová, Eva

    2013-01-01

    The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson’s disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells. PMID:23951389

  20. Chromatin landscape dictates HSF binding to target DNA elements.

    PubMed

    Guertin, Michael J; Lis, John T

    2010-09-09

    Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. We used the ChIP-seq assay to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in "active" chromatin is a primary determinant of whether HSF can bind following heat shock.

  1. Repression and activation by multiprotein complexes that alter chromatin structure.

    PubMed

    Kingston, R E; Bunker, C A; Imbalzano, A N

    1996-04-15

    Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

  2. Detecting ATM-Dependent Chromatin Modification in DNA Damage Response

    PubMed Central

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lope; Hunt, Clayton; Pandita, Tej K.

    2015-01-01

    Loss of function or mutation of the ataxia–telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage. PMID:25827888

  3. Varied interactions between proviruses and adjacent host chromatin.

    PubMed Central

    Conklin, K F; Groudine, M

    1986-01-01

    Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described. Images PMID:3025623

  4. The Chd1 chromatin remodeler shifts hexasomes unidirectionally

    PubMed Central

    Levendosky, Robert F; Sabantsev, Anton; Deindl, Sebastian; Bowman, Gregory D

    2016-01-01

    Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo. DOI: http://dx.doi.org/10.7554/eLife.21356.001 PMID:28032848

  5. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  6. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  7. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

    PubMed

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

  8. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  9. Chromatin domain boundary element search tool for Drosophila

    PubMed Central

    Srinivasan, Arumugam; Mishra, Rakesh K.

    2012-01-01

    Chromatin domain boundary elements prevent inappropriate interaction between distant or closely spaced regulatory elements and restrict enhancers and silencers to correct target promoters. In spite of having such a general role and expected frequent occurrence genome wide, there is no DNA sequence analysis based tool to identify boundary elements. Here, we report chromatin domain Boundary Element Search Tool (cdBEST), to identify boundary elements. cdBEST uses known recognition sequences of boundary interacting proteins and looks for ‘motif clusters’. Using cdBEST, we identified boundary sequences across 12 Drosophila species. Of the 4576 boundary sequences identified in Drosophila melanogaster genome, >170 sequences are repetitive in nature and have sequence homology to transposable elements. Analysis of such sequences across 12 Drosophila genomes showed that the occurrence of repetitive sequences in the context of boundaries is a common feature of drosophilids. We use a variety of genome organization criteria and also experimental test on a subset of the cdBEST boundaries in an enhancer-blocking assay and show that 80% of them indeed function as boundaries in vivo. These observations highlight the role of cdBEST in better understanding of chromatin domain boundaries in Drosophila and setting the stage for comparative analysis of boundaries across closely related species. PMID:22287636

  10. Role of Rb family in the epigenetic definition of chromatin.

    PubMed

    Gonzalo, Susana; Blasco, María A

    2005-06-01

    Epigenetic changes can influence a variety of cellular processes from regulation of gene transcription to proper chromosome segregation. The molecular activities that dictate the assembly, maintenance and regulation of chromatin structure are beginning to be identified. A recent study demonstrates that the Rb family of tumor suppressors plays a major role in global chromatin structure. In addition to the well-known function of Rb family inducing a repressive chromatin state around euchromatic promoters, Rb proteins have a direct role in the assembly of pericentric and telomeric heterochromatin domains. In particular, the Rb family maintains histone 4 lysine 20 tri-methylation (H4K20) at these constitutive heterochromatin domains. Lack of the Rb family results in decreased H4K20 tri-methylation, coincidental with chromosome segregation defects and abnormal telomere elongation, two processes frequently altered in human cancer. Maintenance of heterochromatic domains, such as those of centromeres and telomeres, may represent a novel tumor suppressor function for the Rb family by ensuing genomic stability.

  11. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  12. Topological interactions between ring polymers: Implications for chromatin loops

    NASA Astrophysics Data System (ADS)

    Bohn, Manfred; Heermann, Dieter W.

    2010-01-01

    Chromatin looping is a major epigenetic regulatory mechanism in higher eukaryotes. Besides its role in transcriptional regulation, chromatin loops have been proposed to play a pivotal role in the segregation of entire chromosomes. The detailed topological and entropic forces between loops still remain elusive. Here, we quantitatively determine the potential of mean force between the centers of mass of two ring polymers, i.e., loops. We find that the transition from a linear to a ring polymer induces a strong increase in the entropic repulsion between these two polymers. On top, topological interactions such as the noncatenation constraint further reduce the number of accessible conformations of close-by ring polymers by about 50%, resulting in an additional effective repulsion. Furthermore, the transition from linear to ring polymers displays changes in the conformational and structural properties of the system. In fact, ring polymers adopt a markedly more ordered and aligned state than linear ones. The forces and accompanying changes in shape and alignment between ring polymers suggest an important regulatory function of such a topology in biopolymers. We conjecture that dynamic loop formation in chromatin might act as a versatile control mechanism regulating and maintaining different local states of compaction and order.

  13. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

    PubMed

    Ortega-Atienza, Sara; Wong, Victor C; DeLoughery, Zachary; Luczak, Michal W; Zhitkovich, Anatoly

    2016-01-08

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

  14. Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

    PubMed

    Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

    2017-01-31

    Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

  15. Chromatin accessibility contributes to simultaneous mutations of cancer genes

    PubMed Central

    Shi, Yi; Su, Xian-Bin; He, Kun-Yan; Wu, Bing-Hao; Zhang, Bo-Yu; Han, Ze-Guang

    2016-01-01

    Somatic mutations of many cancer genes tend to co-occur (termed co-mutations) in certain patterns during tumor initiation and progression. However, the genetic and epigenetic mechanisms that contribute to the co-mutations of these cancer genes have yet to be explored. Here, we systematically investigated the association between the somatic co-mutations of cancer genes and high-order chromatin conformation. Significantly, somatic point co-mutations in protein-coding genes were closely associated with high-order spatial chromatin folding. We propose that these regions be termed Spatial Co-mutation Hotspots (SCHs) and report their occurrence in different cancer types. The conserved mutational signatures and DNA sequences flanking these point co-mutations, as well as CTCF-binding sites, are also enriched within the SCH regions. The genetic alterations that are harboured in the same SCHs tend to disrupt cancer driver genes involved in multiple signalling pathways. The present work demonstrates that high-order spatial chromatin organisation may contribute to the somatic co-mutations of certain cancer genes during tumor development. PMID:27762310

  16. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

    NASA Astrophysics Data System (ADS)

    Sanborn, Adrian; Rao, Suhas; Huang, Su-Chen; Durand, Neva; Huntley, Miriam; Jewett, Andrew; Bochkov, Ivan; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher; McKenna, Doug; Stamenova, Elena; Gnirke, Andreas; Melnikov, Alexandre; Lander, Eric; Aiden, Erez

    Our recent kilobase-resolution genome-wide maps of DNA self-contacts demonstrated that mammalian genomes are organized into domains and loops demarcated by the DNA-binding protein CTCF. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that domains are inconsistent with equilibrium and fractal models. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during condensation leads to formation of an anisotropic ``tension globule.'' In the other, CTCF and cohesin act together to extrude unknotted loops. Both models are consistent with the observed domains and loops. However, the extrusion model explains a far wider array of observations, such as why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The extrusion model predicts in silico the experimental maps using only CTCF-binding sites. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

  17. In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication.

    PubMed Central

    Alexiadis, V; Varga-Weisz, P D; Bonte, E; Becker, P B; Gruss, C

    1998-01-01

    DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro. PMID:9628878

  18. Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-06-01

    The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

  19. RNA is an integral component of chromatin that contributes to its structural organization.

    PubMed

    Rodríguez-Campos, Antonio; Azorín, Fernando

    2007-11-14

    Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA(-) and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s) are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with alpha-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

  20. The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

    PubMed Central

    Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

    2014-01-01

    A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

  1. Synapsis, recombination, and chromatin remodeling in the XY body of armadillos.

    PubMed

    Sciurano, Roberta B; Rahn, Mónica I; Rossi, Luis; Luaces, Juan Pablo; Merani, María Susana; Solari, Alberto J

    2012-02-01

    Three xenarthrans species Chaetophractus villosus, Chaetophractus vellerosus, and Zaedyus pichiy have been used for the analysis of the structure, behavior, and immunochemical features of the XY body during pachytene. In all these species, the sex chromosomes form an XY body easily identifiable in thin sections by the special and regular packing of the chromatin fibers of the internal region of the XY body ("differential" regions) and those of the peripheral region (synaptic region). Spermatocyte spreads show a complete synapsis between the X- and the Y-axis, which lasts up to the end of pachytene. From the early pachytene substages to the late ones, the X-axis develops prominent branches, which in late pachytene span the synaptic region. Synapsis is regular as shown by SYCP1 labeling. Axial development is followed by SYCP3 labeling and in the asynaptic region of the X-axis by BRCA1. Gamma-H2AX labels exclusively the differential (asynaptic) region of the X chromosome. A single focus is labeled by MLH1 in the synaptic region. The location of this MLH1 focus spans from 0.3 to 1.6 μm from the telomere in the analyzed xenarthrans, covering approximately half of the Y-axis length. It is concluded that xenarthrans, as basal placental mammals, harbor the largest pseudoautosomal regions of presently analyzed mammals, and shows the typical features of meiotic sex chromosome inactivation (MSCI).

  2. Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells

    PubMed Central

    Shinkai, Soya; Nozaki, Tadasu; Maeshima, Kazuhiro

    2016-01-01

    The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100–500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells. PMID:27764097

  3. Polyimide Fibers

    NASA Technical Reports Server (NTRS)

    St.Clair, Terry L. (Inventor); Fay, Catharine C. (Inventor); Working, Dennis C. (Inventor)

    1997-01-01

    A polyimide fiber having textile physical property characteristics and the process of melt extruding same from a polyimide powder. Polyimide powder formed as the reaction product of the monomers 3.4'-ODA and ODPA, and endcapped with phthalic anhydride to control the molecular weight thereof, is melt extruded in the temperature range of 340? C. to 360? C. and at heights of 100.5 inches, 209 inches and 364.5 inches. The fibers obtained have a diameter in the range of 0.0068 inch to 0.0147 inch; a mean tensile strength in the range of 15.6 to 23.1 ksi; a mean modulus of 406 to 465 ksi; and a mean elongation in the range of 14 to 103%.

  4. Polyimide Fibers

    NASA Technical Reports Server (NTRS)

    St.Clair, Terry L. (Inventor); Fay, Catharine C. (Inventor); Working, Dennis C. (Inventor)

    1998-01-01

    A polyimide fiber having textile physical property characteristics and the process of melt extruding same from a polyimide powder. Polyimide powder formed as the reaction product of the monomers 3.4'-ODA and ODPA, and end- capped with phthalic anhydride to control the molecular weight thereof, is melt extruded in the temperature range of 340 C. to 360 C. and at heights of 100.5 inches. 209 inches and 364.5 inches. The fibers obtained have a diameter in the range of 0.0068 inch to 0.0147 inch; a mean tensile strength in the range of 15.6 to 23.1 ksi; a mean modulus of 406 to 465 ksi, and a mean elongation in the range of 14 to 103%.

  5. Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization.

    PubMed

    Ea, Vuthy; Baudement, Marie-Odile; Lesne, Annick; Forné, Thierry

    2015-07-27

    Recent investigations on 3D chromatin folding revealed that the eukaryote genomes are both highly compartmentalized and extremely dynamic. This review presents the most recent advances in topological domains' organization of the eukaryote genomes and discusses the relationship to chromatin loop formation. CTCF protein appears as a central factor of these two organization levels having either a strong insulating role at TAD borders, or a weaker architectural role in chromatin loop formation. TAD borders directly impact on chromatin dynamics by restricting contacts within specific genomic portions thus confining chromatin loop formation within TADs. We discuss how sub-TAD chromatin dynamics, constrained into a recently described statistical helix conformation, can produce functional interactions by contact stabilization.

  6. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.

    PubMed Central

    Wolffe, A P

    1989-01-01

    Xenopus sperm chromatin lacks class III transcription complexes and somatic histone H1. Inactive class III genes in sperm chromatin are easily programmed with transcription complexes de novo and transcribed in Xenopus oocyte nuclear extract. In contrast, repressed class III genes in somatic chromatin are not transcribed in the oocyte nuclear extract. Class III genes that are initially inactive or repressed in both types of chromatin can be efficiently transcribed in a cell free preparation of Xenopus eggs. Chromatin mediated repression of class III genes in somatic nuclei is reversible in Xenopus egg extract, but not in the oocyte nuclear extract. Any inhibition of transcription attributed to chromatin assembly onto a gene, will therefore depend on the extract in which transcription is assayed. Images PMID:2915929

  7. Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation.

    PubMed

    Keung, Albert J; Bashor, Caleb J; Kiriakov, Szilvia; Collins, James J; Khalil, Ahmad S

    2014-07-03

    The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation and introduces chromatin-based components and systems for synthetic biology and cellular engineering.

  8. Complexity of chromatin folding is captured by the strings and binders switch model.

    PubMed

    Barbieri, Mariano; Chotalia, Mita; Fraser, James; Lavitas, Liron-Mark; Dostie, Josée; Pombo, Ana; Nicodemi, Mario

    2012-10-02

    Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.

  9. Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins

    PubMed Central

    Hajihassan, Zahra; Rabbani-Chadegani, Azra

    2009-01-01

    Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process. PMID:19284573

  10. Low-frequency magnetic field effect on cytoskeleton and chromatin.

    PubMed

    Kroupová, Jana; Bártová, Eva; Fojt, Lukás; Strasák, Ludek; Kozubek, Stanislav; Vetterl, Vladimír

    2007-01-01

    The effect of magnetic fields on the living systems is studied in vivo or in vitro in very broad spectrum of organisms, cells and tissues. The mechanism of their acting is not known until now. We studied low-frequency magnetic field effect on cytoskeleton and on the structure of chromatin in human cells. We used cell line of small lung carcinoma (A549) and the effects of magnetic field on cytoskeleton and higher-order chromatin structure were analyzed 96 h of magnetic field exposure. Magnetic field generated by the cylindrical soil was homogenous and the cells were cultivated at 37 degrees C in humidified atmosphere containing 5% CO(2). Magnetic field induction was B(m)=2 mT and the net frequency f=50 Hz. In such affected and control cells the F-actin was estimated using FITC-conjugated Phalloidin and mitochondria were studied using MitoTracker (Molecular Probes). Images of cytoskeleton and genetic loci were acquired using confocal microscopy and analysis was performed by FISH 2.0 software. Slight morphological changes of F-actin filaments and mitochondria were observed in affected cells and nuclear condensation was found. These effects could be related to the process of cell death apoptosis probably induced by magnetic field. The studies aimed at centromeric heterochromatin (9cen) did not show statistically significant changes. Therefore, we suggest that magnetic field has no influence on higher order chromatin structure but certain changes could be observed on the level of cytoskeleton. However, these statements need a thorough verification. Our preliminary experiments will be extended and the effect of magnetic field on another structures of cytoskeleton and cell nuclei will be further studied.

  11. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

    SciTech Connect

    Finan, John D.; Leddy, Holly A.; Guilak, Farshid

    2011-05-06

    Highlights: {yields} The rate of nucleocytoplasmic transport increases under hyper-osmotic stress. {yields} The mechanism is a change in nuclear geometry, not a change in permeability of the nuclear envelope. {yields} Intracytoplasmic but not intranuclear diffusion is sensitive to osmotic stress. {yields} Pores in the chromatin of the nucleus enlarge under hyper-osmotic stress. -- Abstract: Osmotic stress is a potent regulator of biological function in many cell types, but its mechanism of action is only partially understood. In this study, we examined whether changes in extracellular osmolality can alter chromatin condensation and the rate of nucleocytoplasmic transport, as potential mechanisms by which osmotic stress can act. Transport of 10 kDa dextran was measured both within and between the nucleus and the cytoplasm using two different photobleaching methods. A mathematical model was developed to describe fluorescence recovery via nucleocytoplasmic transport. As osmolality increased, the diffusion coefficient of dextran decreased in the cytoplasm, but not the nucleus. Hyper-osmotic stress decreased nuclear size and increased nuclear lacunarity, indicating that while the nucleus was getting smaller, the pores and channels interdigitating the chromatin had expanded. The rate of nucleocytoplasmic transport was increased under hyper-osmotic stress but was insensitive to hypo-osmotic stress, consistent with the nonlinear osmotic properties of the nucleus. The mechanism of this osmotic sensitivity appears to be a change in the size and geometry of the nucleus, resulting in a shorter effective diffusion distance for the nucleus. These results may explain physical mechanisms by which osmotic stress can influence intracellular signaling pathways that rely on nucleocytoplasmic transport.

  12. Multiple modes of chromatin configuration at natural meiotic recombination hot spots in fission yeast.

    PubMed

    Hirota, Kouji; Steiner, Walter W; Shibata, Takehiko; Ohta, Kunihiro

    2007-11-01

    The ade6-M26 meiotic recombination hot spot of fission yeast is defined by a cyclic AMP-responsive element (CRE)-like heptanucleotide sequence, 5'-ATGACGT-3', which acts as a binding site for the Atf1/Pcr1 heterodimeric transcription factor required for hot spot activation. We previously demonstrated that the local chromatin around the M26 sequence motif alters to exhibit higher sensitivity to micrococcal nuclease before the initiation of meiotic recombination. In this study, we have examined whether or not such alterations in chromatin occur at natural meiotic DNA double-strand break (DSB) sites in Schizosaccharomyces pombe. At one of the most prominent DSB sites, mbs1 (meiotic break site 1), the chromatin structure has a constitutively accessible configuration at or near the DSB sites. The establishment of the open chromatin state and DSB formation are independent of the CRE-binding transcription factor, Atf1. Analysis of the chromatin configuration at CRE-dependent DSB sites revealed both differences from and similarities to mbs1. For example, the tdh1+ locus, which harbors a CRE consensus sequence near the DSB site, shows a meiotically induced open chromatin configuration, similar to ade6-M26. In contrast, the cds1+ locus is similar to mbs1 in that it exhibits a constitutive open configuration. Importantly, Atf1 is required for the open chromatin formation in both tdh1+ and cds1+. These results suggest that CRE-dependent meiotic chromatin changes are intrinsic processes related to DSB formation in fission yeast meiosis. In addition, the results suggest that the chromatin configuration in natural meiotic recombination hot spots can be classified into at least three distinct categories: (i) an Atf1-CRE-independent constitutively open chromatin configuration, (ii) an Atf1-CRE-dependent meiotically induced open chromatin configuration, and (iii) an Atf1-CRE-dependent constitutively open chromatin configuration.

  13. The chromatin landscape of Drosophila: comparisons between species, sexes, and chromosomes.

    PubMed

    Brown, Emily J; Bachtrog, Doris

    2014-07-01

    The chromatin landscape is key for gene regulation, but little is known about how it differs between sexes or between species. Here, we study the sex-specific chromatin landscape of Drosophila miranda, a species with young sex chromosomes, and compare it with Drosophila melanogaster. We analyze six histone modifications in male and female larvae of D. miranda (H3K4me1, H3K4me3, H3K36me3, H4K16ac, H3K27me3, and H3K9me2), and define seven biologically meaningful chromatin states that show different enrichments for transcribed and silent genes, repetitive elements, housekeeping, and tissue-specific genes. The genome-wide distribution of both active and repressive chromatin states differs between males and females. In males, active chromatin is enriched on the X, relative to females, due to dosage compensation of the hemizygous X. Furthermore, a smaller fraction of the euchromatic portion of the genome is in a repressive chromatin state in males relative to females. However, sex-specific chromatin states appear not to explain sex-biased expression of genes. Overall, conservation of chromatin states between male and female D. miranda is comparable to conservation between D. miranda and D. melanogaster, which diverged >30 MY ago. Active chromatin states are more highly conserved across species, while heterochromatin shows very low levels of conservation. Divergence in chromatin profiles contributes to expression divergence between species, with ∼26% of genes in different chromatin states in the two species showing species-specific or species-biased expression, an enrichment of approximately threefold over null expectation. Our data suggest that heteromorphic sex chromosomes in males (that is, a hypertranscribed X and an inactivated Y) may contribute to global redistribution of active and repressive chromatin marks between chromosomes and sexes.

  14. Modification of Chromatin Structure by the Thyroid Hormone Receptor.

    PubMed

    Li; Sachs; Shi; Wolffe

    1999-05-01

    Pioneering experiments and recent observations have established the thyroid hormone receptor as a master manipulator of the chromosomal environment in targeting the activation and repression of transcription. Here we review how the thyroid hormone receptor is assembled into chromatin, where in the absence of thyroid hormone the receptor recruits histone deacetylase to silence transcription. On addition of hormone, the receptor undergoes a conformational change that leads to the release of deacetylase, while facilitating the recruitment of transcriptional coactivators that act as histone acetyltransferases. We discuss the biological importance of these observations for gene control by the thyroid hormone receptor and for oncogenic transformation by the mutated thyroid hormone receptor, v-ErbA.

  15. Sequence analysis of chromatin immunoprecipitation data for transcription factors

    PubMed Central

    Fraenkel, Ernest

    2013-01-01

    Chromatin immunoprecipitation (ChIP) experiments allow the location of transcription factors to be determined across the genome. Subsequent analysis of the sequences of the identified regions allows binding to be localized at a higher resolution than can be achieved by current high-throughput experiments without sequence analysis, and may provide important insight into the regulatory programs enacted by the protein of interest. In this chapter we review the tools, workflow, and common pitfalls of such analyses, and recommend strategies for effective motif discovery from these data. PMID:20827592

  16. Effects of nuclear isolation on psoralen affinity for chromatin

    SciTech Connect

    Inadomi, T.; Ross, P.M. )

    1989-09-29

    We have tested the effects of nuclear isolation on intercalation of TMP (a psoralen) at specific sequences and in total DNA of cultured human cells. DNA in nuclei photobound about 20% more TMP than in cells and about 10% as much as purified DNA. In contrast, a transcribed ras gene and a randomly selected polymorphic sequence each bound about 20% more TMP than total DNA in cells. However, in nuclei, as in purified DNA, both sequences were just as sensitive as total DNA. Apparently, chromatin in cells exists within diverse TMP-binding environments and some of this diversity was lost upon nuclear isolation.

  17. Chromatin Imaging with Time-Lapse Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    Time-lapse atomic force microscopy (AFM) is widely used for direct visualization of the nanoscale dynamics of various biological systems. The advent of high-speed AFM instrumentation made it possible to image the dynamics of proteins and protein-DNA complexes within millisecond time range. This chapter describes protocols for studies of structure and dynamics of nucleosomes with time-lapse AFM including the high-speed AFM instrument. The necessary specifics for the preparation of chromatin samples for imaging with AFM including the protocols for the surface preparation are provided. PMID:25827873

  18. Moving chromatin within the interphase nucleus- controlled transitions?

    PubMed Central

    Chuang, Chien-Hui; Belmont, Andrew S.

    2007-01-01

    The past decade has seen an increasing appreciation for nuclear compartmentalization as an underlying determinant of interphase chromosome nuclear organization. To date, attention has focused primarily on describing differential localization of particular genes or chromosome regions as a function of differentiation, cell cycle position, and/or transcriptional activity. The question of how exactly interphase chromosome compartmentalization is established and in particular how interphase chromosomes might move during changes in nuclear compartmentalization, has received less attention. Here we review what is known concerning chromatin mobility in relationship to physiologically regulated changes in nuclear interphase chromosome organization. PMID:17905613

  19. Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure.

    PubMed

    Szerlong, Heather J; Hansen, Jeffrey C

    2011-02-01

    Genetic information in eukaryotes is managed by strategic hierarchical organization of chromatin structure. Primary chromatin structure describes an unfolded nucleosomal array, often referred to as "beads on a string". Chromatin is compacted by the nonlinear rearrangement of nucleosomes to form stable secondary chromatin structures. Chromatin conformational transitions between primary and secondary structures are mediated by both nucleosome-stacking interactions and the intervening linker DNA. Chromatin model system studies find that the topography of secondary structures is sensitive to the spacing of nucleosomes within an array. Understanding the relationship between nucleosome spacing and higher order chromatin structure will likely yield important insights into the dynamic nature of secondary chromatin structure as it occurs in vivo. Genome-wide nucleosome mapping studies find the distance between nucleosomes varies, and regions of uniformly spaced nucleosomes are often interrupted by regions of nonuniform spacing. This type of organization is found at a subset of actively transcribed genes in which a nucleosome-depleted region near the transcription start site is directly adjacent to uniformly spaced nucleosomes in the coding region. Here, we evaluate secondary chromatin structure and discuss the structural and functional implications of variable nucleosome distributions in different organisms and at gene regulatory junctions.

  20. The role of Nucleosome Positions on Chromatin Structure: A multi-scale approach

    NASA Astrophysics Data System (ADS)

    Lequieu, Joshua; Cordoba, Andres; de Pablo, Juan J.

    Nucleosomes compose the basic unit of chromatin, and their locations are central to the regulation and compaction of eukaryotic genomes. In this work, we examine the coupling between different length scales within chromatin by examining the influence of nucleosome positions on three-dimensional chromatin structure. First, using a detailed molecular model of DNA and proteins, we predict the one-dimensional positioning of nucleosomes and the repositioning mechanisms of nucleosomal DNA. We demonstrate that this mechanism is strongly dependent on DNA sequence and that DNA slides around the histone proteins by either a screw-like or loop-like rearrangement. Next, we couple this detailed model to a coarsened model of chromatin and examine the impact of DNA sequence on chromatin's three-dimensional structure. We show that both the locations of nucleosomes and the mechanisms by which they move have a significant impact on higher-order chromatin structure and that variations in DNA sequence lead to ''open'' or ''closed'' regions of chromatin. This approach represents an efficient tool towards understanding the higher order structure of chromatin and how various aspects of chromatin structure are coupled together.

  1. CHROMATIN ASSEMBLY AND TRANSCRIPTIONAL CROSS-TALK IN XENOPUS LAEVIS OOCYTE AND EGG EXTRACTS

    PubMed Central

    Wang, Wei-Lin; Shechter, David

    2016-01-01

    Chromatin, primarily a complex of DNA and histone proteins, is the physiological form of the genome. Chromatin is generally repressive for transcription and other information transactions that occur on DNA. A wealth of post-translational modifications on canonical histones and histone variants encode regulatory information to recruit or repel effector proteins on chromatin, promoting and further repressing transcription and thereby form the basis of epigenetic information. During metazoan oogenesis, large quantities of histone proteins are synthesized and stored in preparation for the rapid early cell cycles of development and to elicit maternal control of chromatin assembly pathways. Oocyte and egg cell-free extracts of the frog Xenopus laevis are a compelling model system for the study of chromatin assembly and transcription precisely because they exist in an extreme state primed for rapid chromatin assembly or for transcriptional activity. We show that chromatin assembly rates are slower in X. laevis oocyte than in egg extracts, while conversely only oocyte extracts transcribe template plasmids. We demonstrate that rapid chromatin assembly in egg extracts represses RNA Polymerase II dependent transcription, while pre-binding of TATA-Binding Protein (TBP) to a template plasmid promotes transcription. Our experimental evidence presented here supports a model in which chromatin assembly and transcription are in competition and that the onset of zygotic genomic activation may be in part due to stable transcriptional complex assembly. PMID:27759158

  2. Maternal chromatin remodeling during maturation and after fertilization in mouse oocytes.

    PubMed

    Spinaci, Marcella; Seren, Eraldo; Mattioli, Mauro

    2004-10-01

    Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation.

  3. Chromatin poises miRNA- and protein-coding genes for expression.

    PubMed

    Barski, Artem; Jothi, Raja; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Schones, Dustin E; Zhao, Keji

    2009-10-01

    Chromatin modifications have been implicated in the regulation of gene expression. While association of certain modifications with expressed or silent genes has been established, it remains unclear how changes in chromatin environment relate to changes in gene expression. In this article, we used ChIP-seq (chromatin immunoprecipitation with massively parallel sequencing) to analyze the genome-wide changes in chromatin modifications during activation of total human CD4(+) T cells by T-cell receptor (TCR) signaling. Surprisingly, we found that the chromatin modification patterns at many induced and silenced genes are relatively stable during the short-term activation of resting T cells. Active chromatin modifications were already in place for a majority of inducible protein-coding genes, even while the genes were silent in resting cells. Similarly, genes that were silenced upon T-cell activation retained positive chromatin modifications even after being silenced. To investigate if these observations are also valid for miRNA-coding genes, we systematically identified promoters for known miRNA genes using epigenetic marks and profiled their expression patterns using deep sequencing. We found that chromatin modifications can poise miRNA-coding genes as well. Our data suggest that miRNA- and protein-coding genes share similar mechanisms of regulation by chromatin modifications, which poise inducible genes for activation in response to environmental stimuli.

  4. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  5. Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler

    PubMed Central

    Bowman, Gregory D.

    2016-01-01

    ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo. PMID:26993344

  6. Evolution and genetic architecture of chromatin accessibility and function in yeast.

    PubMed

    Connelly, Caitlin F; Wakefield, Jon; Akey, Joshua M

    2014-07-01

    Chromatin accessibility is an important functional genomics phenotype that influences transcription factor binding and gene expression. Genome-scale technologies allow chromatin accessibility to be mapped with high-resolution, facilitating detailed analyses into the genetic architecture and evolution of chromatin structure within and between species. We performed Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-Seq) to map chromatin accessibility in two parental haploid yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus and their diploid hybrid. We show that although broad-scale characteristics of the chromatin landscape are well conserved between these species, accessibility is significantly different for 947 regions upstream of genes that are enriched for GO terms such as intracellular transport and protein localization exhibit. We also develop new statistical methods to investigate the genetic architecture of variation in chromatin accessibility between species, and find that cis effects are more common and of greater magnitude than trans effects. Interestingly, we find that cis and trans effects at individual genes are often negatively correlated, suggesting widespread compensatory evolution to stabilize levels of chromatin accessibility. Finally, we demonstrate that the relationship between chromatin accessibility and gene expression levels is complex, and a significant proportion of differences in chromatin accessibility might be functionally benign.

  7. A critical role for chromatin in mounting a synergistic transcriptional response to GAL4-VP16.

    PubMed Central

    Chang, C; Gralla, J D

    1994-01-01

    The role of chromatin in mounting a synergistic transcriptional response to GAL4-VP16 was investigated. Strong synergy was observed when chromatin templates were used in vitro. The synergy was severely reduced when naked DNA templates were transcribed. In vivo synergy was strong when nonreplicating templates were used. However, the use of replicating templates, which involved transient disruptions of chromatin, led to strong reductions in synergy. In both of these low-synergy responses, transcription levels were high. We infer that strong synergy has a requirement for chromatin that may be understood in terms of the competition between multiple activator molecules and histone cores for promoter DNA. Images PMID:8035798

  8. DNA repeat lengths of erythrocyte chromatins differing in content of histones H1 and H5.

    PubMed Central

    Miki, B L; Neelin, J M

    1980-01-01

    Among the erythrocytes of chicken, trout, carp, and sucker, the relative proportion of the lysine-rich histone H5 varied from 20 to 0% of the total histones. Following digestion of nuclear chromatin with micrococcal nuclease, each of them displayed a longer DNA repeat length and greater repeat length heterogeneity than found in liver chromatin. Fish erythrocytes possessed similar repeat lengths of 207-209 base pairs which was 10-12 base pairs shorter than in chicken erythrocyte chromatin and approximately 10 base pairs longer than in liver chromatin. No correlation existed between the DNA repeat length or repeat length heterogeneity and the relative proportion of H5. Images PMID:6777761

  9. Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

    PubMed

    Mittal, Chitvan; Blacketer, Melissa J; Shogren-Knaak, Michael A

    2014-07-15

    The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.

  10. Probing the impact of chromatin conformation on genome editing tools

    PubMed Central

    Chen, Xiaoyu; Rinsma, Marrit; Janssen, Josephine M.; Liu, Jin; Maggio, Ignazio; Gonçalves, Manuel A.F.V.

    2016-01-01

    Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions. PMID:27280977

  11. Nuclear constriction segregates mobile nuclear proteins away from chromatin

    PubMed Central

    Irianto, Jerome; Pfeifer, Charlotte R.; Bennett, Rachel R.; Xia, Yuntao; Ivanovska, Irena L.; Liu, Andrea J.; Greenberg, Roger A.; Discher, Dennis E.

    2016-01-01

    As a cell squeezes its nucleus through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, chromatin density and nuclear factors could in principle be physically perturbed. Here, in cancer cell migration through rigid micropores and in passive pulling into micropipettes, local compaction of chromatin is observed coincident with depletion of mobile factors. Heterochromatin/euchromatin was previously estimated from molecular mobility measurements to occupy a volume fraction f of roughly two-thirds of the nuclear volume, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g., BRCA1, 53BP1) or nucleases (e.g., Cas9, FokI), are depleted within the constriction, approaching 0%. Such losses—compounded by the occasional rupture of the nuclear envelope—can have important functional consequences. Studies of a nuclease that targets a locus in chromosome-1 indeed show that constricted migration delays DNA damage. PMID:27798234

  12. Molecular Architecture of  Yeast Chromatin Assembly Factor 1

    PubMed Central

    Kim, Daegeun; Setiaputra, Dheva; Jung, Taeyang; Chung, Jaehee; Leitner, Alexander; Yoon, Jungmin; Aebersold, Ruedi; Hebert, Hans; Yip, Calvin K.; Song, Ji-Joon

    2016-01-01

    Chromatin Assembly Complex 1 (CAF-1) is a major histone chaperone involved in deposition of histone H3 and H4 into nucleosome. CAF-1 is composed of three subunits; p150, p60 and p48 for human and Cac1, Cac2 and Cac3 for yeast. Despite of its central role in chromatin formation, structural features of the full CAF-1 in complex with histones and other chaperones have not been well characterized. Here, we dissect molecular architecture of yeast CAF-1 (yCAF-1) by cross-linking mass spectrometry (XL-MS) and negative stain single-particle electron microscopy (EM). Our work revealed that Cac1, the largest subunit of yCAF-1, might serve as a major histone binding platform linking Cac2 and Cac3. In addition, EM analysis showed that yCAF-1 adopts a bilobal shape and Cac1 connecting Cac2 and Cac3 to generate a platform for binding histones. This study provides the first structural glimpse of the full CAF-1 complex and a structural framework to understand histone chaperoning processes. PMID:27221973

  13. Stacking the DEK: From chromatin topology to cancer stem cells

    PubMed Central

    Privette Vinnedge, Lisa M.; Kappes, Ferdinand; Nassar, Nicolas; Wells, Susanne I.

    2013-01-01

    Stem cells are essential for development and tissue maintenance and display molecular markers and functions distinct from those of differentiated cell types in a given tissue. Malignant cells that exhibit stem cell-like activities have been detected in many types of cancers and have been implicated in cancer recurrence and drug resistance. Normal stem cells and cancer stem cells have striking commonalities, including shared cell surface markers and signal transduction pathways responsible for regulating quiescence vs. proliferation, self-renewal, pluripotency and differentiation. As the search continues for markers that distinguish between stem cells, progenitor cells and cancer stem cells, growing evidence suggests that a unique chromatin-associated protein called DEK may confer stem cell-like qualities. Here, we briefly describe current knowledge regarding stem and progenitor cells. We then focus on new findings that implicate DEK as a regulator of stem and progenitor cell qualities, potentially through its unusual functions in the regulation of local or global chromatin organization. PMID:23255114

  14. Coupling Circadian Rhythms of Metabolism and Chromatin Remodeling

    PubMed Central

    Masri, Selma; Orozco-Solis, Ricardo; Aguilar-Arnal, Lorena; Cervantes, Marlene; Sassone-Corsi, Paolo

    2015-01-01

    The circadian clock controls a large variety of neuronal, endocrine, behavioral and physiological responses in mammals. This control is exerted in large part at the transcriptional level on genes expressed in a cyclic manner. A highly specialized transcriptional machinery based on clock regulatory factors organized in feedback autoregulatory loops governs a significant portion of the genome. These oscillations in gene expression are paralleled by critical events of chromatin remodeling that appear to provide plasticity to circadian regulation. Specifically, the NAD+-dependent deacetylases SIRT1 and SIRT6 have been linked to circadian control of gene expression. This, and additional accumulating evidence, shows that the circadian epigenome appears to share intimate links with cellular metabolic processes and has remarkable plasticity showing reprogramming in response to nutritional challenges. In addition to SIRT1 and SIRT6, a number of chromatin remodelers have been implicated in clock control, including the histone H3K4 tri-methyltransferase MLL1. Deciphering the molecular mechanisms that link metabolism, epigenetic control and circadian responses will provide valauble insights towards innovative strategies of therapeutic intervention. PMID:26332964

  15. Analysis of chromatin boundary activity in Drosophila cells

    PubMed Central

    Li, Mo; Belozerov, Vladimir E; Cai, Haini N

    2008-01-01

    Background Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. Results We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function. Conclusion The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements. PMID:19077248

  16. Reactivation of developmentally silenced globin genes by forced chromatin looping

    PubMed Central

    Krivega, Ivan; Breda, Laura; Motta, Irene; Jahn, Kristen S.; Reik, Andreas; Gregory, Philip D.; Rivella, Stefano; Dean, Ann; Blobel, Gerd A.

    2014-01-01

    Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications. PMID:25126789

  17. Roberts syndrome: New evidence supporting an altered metaphase chromatin structure

    SciTech Connect

    Shang, X.M.; Schultz, E.L.; Tonk, V.

    1994-09-01

    Roberts syndrome is a rare autosomal recessive disease clinically manifested in the newborn by mental and growth retardation, tetraphocomelia, and a variety of craniofacial abnormalities. Cell lines derived from RS patients exhibit subtle mutagen hypersensitivity and cytogenetic abnormalities which include random chromosome loss and the splaying of heterochromatic chromosomal regions. The latter, typically detected on C-banded metaphases, has been used prenatally for the diagnosis of RS. To gain further insights into the RS defect, we have examined a number of parameters related to metaphase chromatin structure, with observations as follows. (1) The heterochromatic splaying associated with RS was found to be visible on G- as well as C-banded metaphases. (2) Quantitative evaluations using fluorescence image analysis revealed that RS metaphase chromosomes bind DAPI less efficiently than chromosomes from normal cells. (3) Denaturation of chromosomal DNA with either a C-banding procedure or 70% formamide at 70{degree}C each produced an aberrant hybridization pattern on RS chromosomes in FISH experiments employing biotinylated total human DNA as probe. (4) RS cells exhibited a >3-fold increase in sensitivity to VM-26, a potent inhibitor of topoisomerase II. Collectively, the aforementioned data support the notion that the primary defect in RS results in an altered metaphase chromatin structure.

  18. Synaptic, transcriptional and chromatin genes disrupted in autism.

    PubMed

    De Rubeis, Silvia; He, Xin; Goldberg, Arthur P; Poultney, Christopher S; Samocha, Kaitlin; Cicek, A Erucment; Kou, Yan; Liu, Li; Fromer, Menachem; Walker, Susan; Singh, Tarinder; Klei, Lambertus; Kosmicki, Jack; Shih-Chen, Fu; Aleksic, Branko; Biscaldi, Monica; Bolton, Patrick F; Brownfeld, Jessica M; Cai, Jinlu; Campbell, Nicholas G; Carracedo, Angel; Chahrour, Maria H; Chiocchetti, Andreas G; Coon, Hilary; Crawford, Emily L; Curran, Sarah R; Dawson, Geraldine; Duketis, Eftichia; Fernandez, Bridget A; Gallagher, Louise; Geller, Evan; Guter, Stephen J; Hill, R Sean; Ionita-Laza, Juliana; Jimenz Gonzalez, Patricia; Kilpinen, Helena; Klauck, Sabine M; Kolevzon, Alexander; Lee, Irene; Lei, Irene; Lei, Jing; Lehtimäki, Terho; Lin, Chiao-Feng; Ma'ayan, Avi; Marshall, Christian R; McInnes, Alison L; Neale, Benjamin; Owen, Michael J; Ozaki, Noriio; Parellada, Mara; Parr, Jeremy R; Purcell, Shaun; Puura, Kaija; Rajagopalan, Deepthi; Rehnström, Karola; Reichenberg, Abraham; Sabo, Aniko; Sachse, Michael; Sanders, Stephan J; Schafer, Chad; Schulte-Rüther, Martin; Skuse, David; Stevens, Christine; Szatmari, Peter; Tammimies, Kristiina; Valladares, Otto; Voran, Annette; Li-San, Wang; Weiss, Lauren A; Willsey, A Jeremy; Yu, Timothy W; Yuen, Ryan K C; Cook, Edwin H; Freitag, Christine M; Gill, Michael; Hultman, Christina M; Lehner, Thomas; Palotie, Aaarno; Schellenberg, Gerard D; Sklar, Pamela; State, Matthew W; Sutcliffe, James S; Walsh, Christiopher A; Scherer, Stephen W; Zwick, Michael E; Barett, Jeffrey C; Cutler, David J; Roeder, Kathryn; Devlin, Bernie; Daly, Mark J; Buxbaum, Joseph D

    2014-11-13

    The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

  19. Surface enhanced Raman spectroscopy on the tip of a plastic optical fiber

    NASA Astrophysics Data System (ADS)

    Taguenang, J. M.; Kassu, A.; Sharma, A.; Diggs, D.

    2007-09-01

    Surface Enhanced Raman Spectroscopy is a powerful analytical technique capable of single molecule detection sensitivity. We have detected SERS on the tip of a 3 mm-core diameter PMMA plastic optical fiber. The technique involves deposition of 30 nm gold nanoparticles followed by deposition of sample of interest to be analyzed. SERS enhancement has been demonstrated for several chemicals like glycerin and dye Rhodamine 6G as well biological molecules like Acetaminophen, aspirin and Streptavidin and poly-L-Lysine. It is shown that interfering spectrum of PMMA can be subtracted to reveal the SERS spectrum of molecule of interest. The technique can simplify SERS detection by connecting the other end of fiber directly to a spectrometer. SERS was recorded for various concentrations of analytes. Using a focused 633 nm laser, a detection sensitivity of 0.1picogram was established.

  20. Simple and efficient L-band erbium-doped fiber amplifiers for WDM networks

    NASA Astrophysics Data System (ADS)

    Choi, H. B.; Oh, J. M.; Lee, D.; Ahn, S. J.; Park, B. S.; Lee, S. B.

    2002-11-01

    The performance of L-band erbium-doped fiber amplifier (EDFA) of a simple structure with a fiber Bragg grating (FBG) was investigated. The injected C-band ASE by the FBG offers low-cost amplification and greatly improves the efficiency of the EDFA. There are 9 and 4 dB improvements with the FBG at 1587 nm, at low and high input, respectively. The flat gain of 18 dB, up to a total input of -5 dBm at 150 mW of 980 nm pump, is obtained over 30 nm with less than ±0.5 dB gain variations without any gain equalizer. The proposed EDFA provides a cost-effective solution for wavelength division multiplexing systems.

  1. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.

    PubMed

    Boettiger, Alistair N; Bintu, Bogdan; Moffitt, Jeffrey R; Wang, Siyuan; Beliveau, Brian J; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A; Wu, Chao-ting; Zhuang, Xiaowei

    2016-01-21

    Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly

  2. The Drosophila Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics In Vivo

    PubMed Central

    Fasulo, Barbara; Deuring, Renate; Murawska, Magdalena; Gause, Maria; Dorighi, Kristel M.; Schaaf, Cheri A.; Dorsett, Dale; Brehm, Alexander; Tamkun, John W.

    2012-01-01

    dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells. PMID:22912596

  3. Carbon-fiber technology

    NASA Technical Reports Server (NTRS)

    Hansen, C. F.; Parker, J. A.

    1980-01-01

    The state of the art of PAN based carbon fiber manufacture and the science of fiber behavior is surveyed. A review is given of the stabilization by oxidation and the subsequent carbonization of fibers, of the apparent structure of fibers deduced from scanning electron microscopy, from X-ray scattering, and from similarities with soft carbons, and of the known relations between fiber properties and heat treatment temperature. A simplified model is invoked to explain the electrical properties of fibers and recent quantum chemical calculations on atomic clusters are used to elucidate some aspects of fiber conductivity. Some effects of intercalation and oxidative modification of finished fibers are summarized.

  4. Chromatin and epigenetics in all their states: Meeting report of the first conference on Epigenetic and Chromatin Regulation of Plant Traits - January 14 - 15, 2016 - Strasbourg, France.

    PubMed

    Bey, Till; Jamge, Suraj; Klemme, Sonja; Komar, Dorota Natalia; Le Gall, Sabine; Mikulski, Pawel; Schmidt, Martin; Zicola, Johan; Berr, Alexandre

    2016-08-02

    In January 2016, the first Epigenetic and Chromatin Regulation of Plant Traits conference was held in Strasbourg, France. An all-star lineup of speakers, a packed audience of 130 participants from over 20 countries, and a friendly scientific atmosphere contributed to make this conference a meeting to remember. In this article we summarize some of the new insights into chromatin, epigenetics, and epigenomics research and highlight nascent ideas and emerging concepts in this exciting area of research.

  5. Next-generation wideband multimode fibers for data centers

    NASA Astrophysics Data System (ADS)

    Balemarthy, Kasyapa; Shubochkin, Roman; Sun, Yi

    2016-03-01

    Short-reach optical links such as those used in data centers pre-dominantly employ VCSELs together with laser- optimized OM4 and OM3 multimode fiber (MMF), mainly due to their reliability, energy-efficiency and low end-to-end system cost. The IEEE 802.3bm specification for 100Gbps Ethernet utilizes four parallel MMFs each operating at a serial data rate of 25Gbps. Due to the rapidly increasing internet traffic, the IEEE P802.3bs Task Force is working towards a 400Gbps Ethernet standard requiring a commensurate increase in the number of parallel fibers deployed. Using 16 parallel lanes, while feasible, is not the most efficient use of cabling. One solution to the data rate - cable density problem is the use of shortwave wavelength division multiplexing (SWDM) near 850nm. For example, employing four wavelengths separated by ~30nm (with an operational window of ~840-950nm) results in a four-fold increase in the per-fiber data rate. Furthermore, SWDM can be combined with the parallel solution to support 400Gbps with the same cable density as the current 100Gbps Ethernet solution using OM4 fiber. Conventional laser-optimized OM4 gives diminished performance at the longer wavelengths compared to 850nm. Shifting the OM4 optimization wavelength to longer wavelengths sacrifices the 850nm performance. In this paper, we present next-generation wideband multimode fibers (NG-WBMMF) that are optimized for SWDM operation using a novel design approach employing multiple dopants. We have fabricated and characterized a wideband MMF that is OM4 compliant over the 850-950nm wavelength window. BER measurements demonstrate that this next-generation WB MMF satisfies the pre-FEC requirement of 5 × 10-5 even after transmission over 300m.

  6. Resistance of the nucleosomal organization of eucaryotic chromatin to ionizing radiation. [/sup 60/Co

    SciTech Connect

    Chiu, S.M.; Oleinick, N.L.

    1982-09-01

    The structural organization and radiation sensitivity of Tetrahymena chromatin under several conditions of modified transcriptional activity were investigated using the structure-specific nucleases, micrococcal nuclease and DNase I. Digestion of unirradiated nuclei by those nucleases proceeded with very similar kinetics and to a similar extent irrespective of the stages of growth of the cultures, except for the cultures in stationary phase, which became more resistant to DNase I digestion. Neither for suppression of total cellular RNA synthesis by actinomycin D nor the transient inhibition of only rRNA synthesis by 40 krad of ..gamma.. radiation affected the sensitivity of the chromatin of the nucleases. These results confirm that activity transcribing chromatin remains in an active conformation even when its function is temporarily inhibited, while more permanent repression of some genes during stationary phase appears to alter the chromatin and hence its susceptibility to DNase I. Actively transcribing ribosomal chromatin was found to be very sensitive to DNase I degradation compared to bulk chromatin; its sensitivity to DNase I was also not altered by 40 krad of ..gamma.. radiation, but was reduced in stationary phase. It is concluded that damage to DNA and/or chromatin resulting from ..gamma.. irradiation does not produce alterations in the nucleosome-level organization of chromatin which can be measured by micrococcal nuclease and DNase I.

  7. ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing.

    PubMed

    Gillespie, Peter J; Khoudoli, Guennadi A; Stewart, Graeme; Swedlow, Jason R; Blow, J Julian

    2007-10-09

    Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1-4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.

  8. Conformational study of the binding of a high mobility group protein with chromatin

    SciTech Connect

    Sasi, R.; Huvoes, P.E.; Fasman, G.D.

    1982-10-10

    The nature of the binding of a high mobility group protein (HMG 17) to native and H1-H5-depleted chicken erythrocyte chromatin was studied, as a function of ionic strength, using circular dichroism and thermal denaturation techniques. The circular dichroism properties of the HMG 17-reconstituted whole chromatin and H1-H5-depleted chromatin structure occurred upon HMG 17 binding at low ionic strength. Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. These results indicate that the binding sites of HMG 17 are situated in the linker regions immediately adjacent to the core. The nature of the interaction of HMG 17 at higher ionic strength with whole chromatin and H1-H5-depleted chromatin was found to be different. These observations suggest that HMG 17 does not loosen chromatin structure but produces an overall stabilization and condensation of structure. The implications of these results to the currently accepted models of transcriptionally active chromatin are discussed.

  9. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  10. ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters

    PubMed Central

    Bailey, Swneke D.; Zhang, Xiaoyang; Desai, Kinjal; Aid, Malika; Corradin, Olivia; Cowper-Sal·lari, Richard; Akhtar-Zaidi, Batool; Scacheri, Peter C.; Haibe-Kains, Benjamin; Lupien, Mathieu

    2015-01-01

    Chromatin interactions connect distal regulatory elements to target gene promoters guiding stimulus- and lineage-specific transcription. Few factors securing chromatin interactions have so far been identified. Here by integrating chromatin interaction maps with the large collection of transcription factor binding profiles provided by the ENCODE project, we demonstrate that the zinc-finger protein ZNF143 preferentially occupies anchors of chromatin interactions connecting promoters with distal regulatory elements. It binds directly to promoters and associates with lineage-specific chromatin interactions and gene expression. Silencing ZNF143 or modulating its DNA-binding affinity using single nucleotide polymorphisms (SNPs) as a surrogate of site-directed mutagenesis reveals the sequence dependency of chromatin interactions at gene promoters. We also find that chromatin interactions alone do not regulate gene expression. Together, our results identify ZNF143 as a novel chromatin-looping factor that contributes to the architectural foundation of the genome by providing sequence specificity at promoters connected with distal regulatory elements. PMID:25645053

  11. Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein, MENT.

    PubMed

    Istomina, Natalia E; Shushanov, Sain S; Springhetti, Evelyn M; Karpov, Vadim L; Krasheninnikov, Igor A; Stevens, Kimberly; Zaret, Kenneth S; Singh, Prim B; Grigoryev, Sergei A

    2003-09-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.

  12. Mechanism of Transcriptional Regulation by Androgen Receptor and its Coactivators in the Context of Chromatin

    DTIC Science & Technology

    2002-07-01

    acetylation over the promoter region was also observed. This histone acetylation is correlated with the recruitment of CBP/p300. Taken together, our...data suggest that hormone-dependent activation by AR is associated with two types of chromatin remodeling, histone acetylation and chromatin remodeling...number of transcriptional cofactors as histone acetyltransferaseases (HAT), deacetylases, or methyltransferases. Acetylation of histone tails is

  13. Mechanism of the Interaction of Plant Alkaloid Vincristine with DNA and Chromatin: Spectroscopic Study

    PubMed Central

    Mohammadgholi, Azadeh; Fallah, Sodabeh

    2013-01-01

    Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA–cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus. PMID:23590199

  14. Mechanism of the interaction of plant alkaloid vincristine with DNA and chromatin: spectroscopic study.

    PubMed

    Mohammadgholi, Azadeh; Rabbani-Chadegani, Azra; Fallah, Sodabeh

    2013-05-01

    Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA-cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus.

  15. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    SciTech Connect

    Persson, Jenna; Ekwall, Karl

    2010-05-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  16. Arabidopsis seed germination responses to osmotic stress involve the chromatin modifier PICKLE.

    PubMed

    Belin, Christophe; Lopez-Molina, Luis

    2008-07-01

    pkl mutant seed germination is hypersensitive to ABA treatment due to abnormally high and persistent ABI3 and ABI5 expression. PKL, a putative chromatin modifier, is instrumental to associate ABI3 and ABI5 with silent chromatin in response to ABA. Thus, PKL prevents exaggerated germination arrest responses by shutting off ABI3 and ABI5 expression in response to mild stresses.

  17. Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin.

    PubMed

    Goffin, C; Verly, W G

    1982-10-01

    Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.

  18. Significance of higher-order chromatin architecture for neuronal function and dysfunction.

    PubMed

    Wilczynski, Grzegorz M

    2014-05-01

    Recent studies in neurons indicate that the large-scale chromatin architectural framework, including chromosome territories or lamina-associated chromatin, undergoes dynamic changes that represent an emergent level of regulation of neuronal gene-expression. This phenomenon has been implicated in neuronal differentiation, long-term potentiation, seizures, and disorders of neural plasticity such as Rett syndrome and epilepsy.

  19. Gibberellin-induced change in the structure of chromatin in wheat sprouts: decrease in the accessibility of DNA in preparations of soluble chromatin to the action of EcoRII methylase

    SciTech Connect

    Noskov, V.A.; Kintsurashvili, L.N.; Smirnova, T.A.; Manamsh'yan, T.A.; Kir'yanov, G.I.; Vanyushin, B.F.

    1986-05-20

    A method has been perfected for producing soluble chromatin from whole wheat sprouts at low ionic strength. The chromatin preparations isolated possess a native structure: they have a nucleosome organization. Under identical conditions the soluble wheat chromatin undergoes more profound degradation by DNase I and staphylococcal nuclease than the chromatin from the rat liver. The DNA contained in the isolated chromatin is capable of accepting CHnumber groups from S-(methyl-/sup 3/H)-adenosylmethionine during incubation with DNA methylase EcoRII; not all the CC A/T GG sequences in DNA are methylated in vivo. Chromatin from gibberellin A/sub 3/-treated wheat sprout DNA accepts 40% fewer CH/sub 3/ groups than that from the control sprouts, which is probably due to the greater compactness of the chromatin. In the case of longer incubation, the level of methylation of the chromatin falls, which may be associated with the presence of DNA-demethylating activity.

  20. High-resolution, genome-wide mapping of chromatin modifications by GMAT.

    PubMed

    Roh, Tae-Young; Zhao, Keji

    2008-01-01

    One major postgenomic challenge is to characterize the epigenomes that control genome functions. The epigenomes are mainly defined by the specific association of nonhistone proteins with chromatin and the covalent modifications of chromatin, including DNA methylation and posttranslational histone modifications. The in vivo protein-binding and chromatin-modification patterns can be revealed by the chromatin immunoprecipitation assay (ChIP). By combining the ChIP assays and the serial analysis of gene expression (SAGE) protocols, we have developed an unbiased and high-resolution genome-wide mapping technique (GMAT) to determine the genome-wide protein-targeting and chromatin-modification patterns. GMAT has been successfully applied to mapping the target sites of the histone acetyltransferase, Gcn5p, in yeast and to the discovery of the histone acetylation islands as an epigenetic mark for functional regulatory elements in the human genome.

  1. Three-dimensional structure of human chromatin accessibility complex hCHRAC by electron microscopy

    SciTech Connect

    Hu, M.; Hainfeld, J.; Zhang, Y.-B.; Qian, L.; Brinas, R. P.; Kuznetsova, L.

    2008-12-01

    ATP-dependent chromatin remodeling complexes modulate the dynamic assembly and remodeling of chromatin involved in DNA transcription, replication, and repair. There is little structural detail known about these important multiple-subunit enzymes that catalyze chromatin remodeling processes. Here we report a three-dimensional structure of the human chromatin accessibility complex, hCHRAC, using single particle reconstruction by negative stain electron microscopy. This structure shows an asymmetric 15 x 10 x 12 nm disk shape with several lobes protruding out of its surfaces. Based on the factors of larger contact area, smaller steric hindrance, and direct involvement of hCHRAC in interactions with the nucleosome, we propose that four lobes on one side form a multiple-site contact surface 10 nm in diameter for nucleosome binding. This work provides the first determination of the three-dimensional structure of the ISWI-family of chromatin remodeling complexes.

  2. High resolution genome-wide mapping of the primary structure of chromatin

    PubMed Central

    Zhang, Zhenhai; Pugh, B. Franklin

    2011-01-01

    The genomic organization of chromatin is increasingly recognized as a key regulator of cell behavior, but deciphering its regulation mechanisms requires detailed knowledge of chromatin’s primary structure - the assembly of nucleosomes throughout the genome. This Primer explains the principles for mapping and analyzing the primary organization of chromatin on a genomic scale. After introducing chromatin organization and its impact on gene regulation and human health, we then describe methods that detect nucleosome positioning and occupancy levels using chromatin-immunoprecipitation in combination with deep sequencing (ChIP-Seq), a strategy that is now straightforward and cost-efficient. We then explore current strategies for converting the sequence information into knowledge about chromatin, an exciting challenge for biologists and bioinformaticians. PMID:21241889

  3. Genome-wide chromatin remodeling modulates the Alu heat shock response.

    PubMed

    Kim, C; Rubin, C M; Schmid, C W

    2001-10-03

    During heat shock recovery in Hela cells, the level of Alu RNA transiently increases with kinetics that approximately parallel the transient expression of heat shock protein mRNAs. Coincidentally, there is a transient increase in the accessibility of Alu chromatin to restriction enzyme cleavage suggesting that an opening and re-closing of chromatin regulates the Alu stress response. Similar changes occur in alpha satellite and LINE1 chromatin showing that heat shock induces a genome-wide remodeling of chromatin structure which is independent of transcription. The increased accessibility of restriction sites within these repetitive sequences is inconsistent with a simple lengthening of the nucleosome linker region but instead suggests a scrambling of nucleosome positions. Chromatin structure and its dynamics account for many of the principal features of SINE transcriptional regulation potentially providing a functional rationale for the dispersion and high copy number of SINEs.

  4. Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus

    PubMed Central

    Hinde, Elizabeth; Kong, Xiangduo; Yokomori, Kyoko; Gratton, Enrico

    2014-01-01

    Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control. PMID:24988341

  5. Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

    PubMed Central

    Stephens, Andrew D.; Haggerty, Rachel A.; Vasquez, Paula A.; Vicci, Leandra; Snider, Chloe E.; Shi, Fu; Quammen, Cory; Mullins, Christopher; Haase, Julian; Taylor, Russell M.; Verdaasdonk, Jolien S.; Falvo, Michael R.; Jin, Yuan; Forest, M. Gregory

    2013-01-01

    The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes. PMID:23509068

  6. Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro

    PubMed Central

    Francis, Nicole J.; Follmer, Nicole E.; Simon, Matthew D.; Aghia, George; Butler, Jeffrey D.

    2009-01-01

    Summary The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin associated proteins and histone modifications. PRC1-class Polycomb Group protein complexes are essential for development, and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. We find that when PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division. PMID:19303136

  7. A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development

    PubMed Central

    Paige, Sharon L.; Thomas, Sean; Stoick-Cooper, Cristi L.; Wang, Hao; Maves, Lisa; Sandstrom, Richard; Pabon, Lil; Reinecke, Hans; Pratt, Gabriel; Keller, Gordon; Moon, Randall T.; Stamatoyannopoulos, John; Murry, Charles E.

    2012-01-01

    Summary Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. PMID:22981225

  8. Gene activation and cell fate control in plants: a chromatin perspective.

    PubMed

    Engelhorn, Julia; Blanvillain, Robert; Carles, Cristel C

    2014-08-01

    In plants, environment-adaptable organogenesis extends throughout the lifespan, and iterative development requires repetitive rounds of activation and repression of several sets of genes. Eukaryotic genome compaction into chromatin forms a physical barrier for transcription; therefore, induction of gene expression requires alteration in chromatin structure. One of the present great challenges in molecular and developmental biology is to understand how chromatin is brought from a repressive to permissive state on specific loci and in a very specific cluster of cells, as well as how this state is further maintained and propagated through time and cell division in a cell lineage. In this review, we report recent discoveries implementing our knowledge on chromatin dynamics that modulate developmental gene expression. We also discuss how new data sets highlight plant specificities, likely reflecting requirement for a highly dynamic chromatin.

  9. Chromatin: Its history, current research, and the seminal researchers and their philosophy.

    PubMed

    Deichmann, Ute

    2015-01-01

    The concept of chromatin as a complex of nucleic acid and proteins in the cell nucleus was developed by cytologists and biochemists in the late 19th century. It was the starting point for biochemical research on DNA and nuclear proteins. Although interest in chromatin declined rapidly at the beginning of the 20th century, a few decades later a new focus on chromatin emerged, which was not only related to its structure, but also to its function in gene regulatory processes in the development of higher organisms. Since the late 20th century, research on chromatin modifications has also been conducted under the label of epigenetics. This article highlights the major phases of chromatin research until the present time and introduces major investigators and their scientific and philosophical outlooks.

  10. New insights into the mechanisms of mammalian erythroid chromatin condensation and enucleation.

    PubMed

    Ji, Peng

    2015-01-01

    A unique feature in mammalian erythropoiesis is the dramatic chromatin condensation followed by enucleation. This step-by-step process starts at the beginning of terminal erythropoiesis after the hematopoietic stem cells are committed to erythroid lineage. Although this phenomenon is known for decades, the mechanisms of chromatin condensation and enucleation remain elusive. Recent advances in cell and molecular biology have started to reveal the molecular pathways in the regulation of chromatin condensation, the establishment of nuclear polarity prior enucleation, and the rearrangement of actin cytoskeleton in enucleation. However, many challenging questions, especially whether and how the apoptotic mechanisms are involved in chromatin condensation and how to dissect the functions of many actin cytoskeleton proteins in cytokinesis and enucleation, remain to be answered. Here I review our current understanding of mammalian erythroid chromatin condensation and enucleation during terminal differentiation with a focus on more recent studies. I conclude with my perspective of future works in this rising topic in developmental and cell biology.

  11. The tethering of chromatin to the nuclear envelope supports nuclear mechanics

    PubMed Central

    Schreiner, Sarah M.; Koo, Peter K.; Zhao, Yao; Mochrie, Simon G. J.; King, Megan C.

    2015-01-01

    The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins. Using novel imaging analyses, we observe that untethering chromatin from the inner nuclear membrane results in highly deformable nuclei in vivo, particularly in response to cytoskeletal forces. Using optical tweezers, we find that isolated nuclei lacking inner nuclear membrane tethers are less stiff than wild-type nuclei and exhibit increased chromatin flow, particularly in frequency ranges that recapitulate the kinetics of cytoskeletal dynamics. We suggest that modulating chromatin flow can define both transient and long-lived changes in nuclear shape that are biologically important and may be altered in disease. PMID:26074052

  12. The Spectrum of Anti-Chromatin/Nucleosome Autoantibodies: Independent and Interdependent Biomarkers of Disease

    PubMed Central

    Mehra, Sonal; Fritzler, Marvin J.

    2014-01-01

    Autoantibodies directed to chromatin components date back to the discovery of the LE cell and the LE cell phenomenon circa 1950, and subsequent evidence that major components of that reaction were chromatin components and histones in particular. Over time, immunoassays ranging from ELISA and line immunoassays to more modern bead-based assays incorporated histone and DNA mixtures, purified histones, and purified nucleosomes leading to a more thorough understanding of the genesis and pathogenetic relationships of antibodies to chromatin components in systemic lupus erythematosus and other autoimmune conditions. More recently, interest has focussed on other components of chromatin such as high mobility group (HMG) proteins both as targets of B cell responses and pro-inflammatory mediators. This review will focus on immunoassays that utilize chromatin components, their clinical relationships, and newer evidence implicating HMG proteins and DNA neutrophil extracellular traps (NETs) as important players in systemic autoimmune rheumatic diseases. PMID:24804269

  13. Transcriptional and Chromatin Regulation during Fasting - The Genomic Era.

    PubMed

    Goldstein, Ido; Hager, Gordon L

    2015-12-01

    An elaborate metabolic response to fasting is orchestrated by the liver and is heavily reliant on transcriptional regulation. In response to hormones (glucagon, glucocorticoids) many transcription factors (TFs) are activated and regulate various genes involved in metabolic pathways aimed at restoring homeostasis: gluconeogenesis, fatty acid oxidation, ketogenesis, and amino acid shuttling. We summarize recent discoveries regarding fasting-related TFs with an emphasis on genome-wide binding patterns. Collectively, the findings we discuss reveal a large degree of cooperation between TFs during fasting that occurs at motif-rich DNA sites bound by a combination of TFs. These new findings implicate transcriptional and chromatin regulation as major determinants of the response to fasting and unravels the complex, multi-TF nature of this response.

  14. Chromatin Dynamics in Vivo: A Game of Musical Chairs

    PubMed Central

    Melters, Daniël P.; Nye, Jonathan; Zhao, Haiqing; Dalal, Yamini

    2015-01-01

    Histones are a major component of chromatin, the nucleoprotein complex fundamental to regulating transcription, facilitating cell division, and maintaining genome integrity in almost all eukaryotes. In addition to canonical, replication-dependent histones, replication-independent histone variants exist in most eukaryotes. In recent years, steady progress has been made in understanding how histone variants assemble, their involvement in development, mitosis, transcription, and genome repair. In this review, we will focus on the localization of the major histone variants H3.3, CENP-A, H2A.Z, and macroH2A, as well as how these variants have evolved, their structural differences, and their functional significance in vivo. PMID:26262644

  15. The Chromatin Signature of Pluripotency: Establishment and Maintenance.

    PubMed

    Di Giammartino, Dafne Campigli; Apostolou, Effie

    The revolutionary discovery that somatic cells can be reprogrammed by a defined set transcription factors to induced pluripotent stem cells (iPSCs) changed dramatically the way we perceive cell fate determination. Importantly, iPSCs, similar to embryo-derived stem cells (ESCs), are characterized by a remarkable developmental plasticity and the capacity to self-renew "indefinitely" under appropriate culture conditions, opening new avenues for personalized therapy and disease modeling. Elucidating the molecular mechanisms that maintain, induce, or alter stem cell identity is crucial for a deeper understanding of cell fate determination and potential translational applications. Intense research over the last 10 years exploiting technological advances in epigenomics and genome editing has unraveled many of the mysteries of pluripotent identity enabling novel and efficient ways to manipulate it for biomedical purposes. In this review, we focus on the chromatin and epigenetic characteristics that distinguish stem cells from somatic cells and their dynamic changes during differentiation and reprogramming.

  16. Chromatin and beyond: the multitasking roles for SIRT6

    PubMed Central

    Kugel, Sita; Mostoslavsky, Raul

    2014-01-01

    In recent years there has been a large expansion in our understanding of SIRT6 biology, including its structure, regulation, biochemical activity and biological roles. SIRT6 functions as an ADP-ribosylase and NAD+-dependent deacylase of both acetyl groups and long-chain fatty acyl groups. Through these functions SIRT6 impacts cellular homeostasis by regulating DNA repair, telomere maintenance and glucose and lipid metabolism, thus affecting diseases such diabetes, obesity, heart disease, and cancer. Such roles may contribute to overall longevity and health of the organism. Until recently, much of the known functions of SIRT6 were restricted to the chromatin. In this article, we seek to describe and expand this knowledge with recent advances in understanding the mechanisms of SIRT6 action and their implications for human biology and disease. PMID:24438746

  17. Anti-aging peptide bioregulators induce reactivation of chromatin.

    PubMed

    Lezhava, T; Monaselidze, J; Kadotani, T; Dvalishvili, N; Buadze, T

    2006-04-01

    The effect of synthetic peptide bioregulators (Epitalon, Livagen and Vilon) on structural and facultative heterochromatin of cultivated lymphocytes have been studied among old (75-88yr.) people. The data obtained indicate that epitalon, livagen and vilon: 1) activate synthetic processes, caused by reactivation of ribosomal genes as a result of deheterochromatinization (decondensation) of nucleolus organizer regions; 2) induce unrolling (deheterochromatinization) of total heterochromatin; 3) release genes repressed by heterochromatinization (condensation) of euchromatic regions forming facultative heterochromatin; 4) epitalon and livagen induce deheterochromatinization (decondensation) of pericentromeric structural heterochromatin of the chromosomes1 and 9. However, vilon does not induce deheterochromatinization of pericentromeric structural heterochromatin. These results indicate that peptide bioregulators Epitalon, Livagen and Vilon cause activation (deheterochromatinization) of chromatin in lymphocytes of old individuals.

  18. Mutations in chromatin machinery and pediatric high-grade glioma

    PubMed Central

    Lulla, Rishi R.; Saratsis, Amanda Muhs; Hashizume, Rintaro

    2016-01-01

    Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors. PMID:27034984

  19. Formation of mammalian erythrocytes: chromatin condensation and enucleation.

    PubMed

    Ji, Peng; Murata-Hori, Maki; Lodish, Harvey F

    2011-07-01

    In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast chromatin condensation and enucleation, and conclude with our perspectives on future studies.

  20. [Effect of irradiation on the degradation of rat thymocyte chromatin].

    PubMed

    Tsudzevich, B O; Parkhomets', Iu P; Andriĭchuk, T R; Iurkina, V V

    1998-01-01

    Genome instability of adaptive nature is formed under the experimental influence on a cell. Under critical conditions, strategy of organism is to damage the cells that cannot be restored and controlled by including the program of apoptosis. The ordered internucleosomal DNA degradation is considered to be one of the proof attributes of immunocompetent cell apoptosis. We investigated the effects of various doses of irradiation on the thymocytes chromatine fragmentation in 1,2,3 hours after a single X-ray exposure or after chronic influence in conditions of Chernobyl research base. By the means of electrophoresis in agarose and judging by polydeoxyribonucleotides accumulation we observed the "ladder pattern" of degradation in 3 hr after single 1 Gr irradiation (the smallest dose displaying the effect). We suppose that the influence of both chronic low-intensity irradiation taking place in Chernobyl and single X-ray exposure result in intensifying of DNA fragmentation in the cells of immunocompetent organs.

  1. Time-resolved spectroscopy and fluorescence resonance energy transfer in the study of excimer laser damage of chromatin

    NASA Astrophysics Data System (ADS)

    Radu, L.; Mihailescu, I.; Radu, S.; Gazdaru, D.

    2007-09-01

    The analysis of chromatin damage produced by a 248 nm excimer laser radiation, for doses of 0.3-3 MJ/m 2 was carried out by time-resolved spectroscopy and fluorescence resonance energy transfer (FRET). The chromatin was extracted from a normal and a tumoral tissue of Wistar rats. The decrease with laser dose of the relative contribution of the excited state lifetimes of ethidium bromide (EtBr) bounded to chromatin constitutes an evidence of the reduction of chromatin deoxyribonucleic acid (DNA) double-strand structure. FRET was performed from dansyl chloride to acridine orange, both coupled to chromatin. The increase of the average distance between these ligands, under the action of laser radiation, reflects a loosening of the chromatin structure. The radiosensitivity of tumor tissue chromatin is higher than that of a normal tissue. The determination of the chromatin structure modification in an excimer laser field can be of interest in laser therapy.

  2. Extensive Transcriptional Regulation of Chromatin Modifiers during Human Neurodevelopment

    PubMed Central

    Weng, Matthias K.; Zimmer, Bastian; Pöltl, Dominik; Broeg, Marc P.; Ivanova, Violeta; Gaspar, John A.; Sachinidis, Agapios; Wüllner, Ullrich

    2012-01-01

    Epigenetic changes, including histone modifications or chromatin remodeling are regulated by a large number of human genes. We developed a strategy to study the coordinate regulation of such genes, and to compare different cell populations or tissues. A set of 150 genes, comprising different classes of epigenetic modifiers was compiled. This new tool was used initially to characterize changes during the differentiation of human embryonic stem cells (hESC) to central nervous system neuroectoderm progenitors (NEP). qPCR analysis showed that more than 60% of the examined transcripts were regulated, and >10% of them had a >5-fold increased expression. For comparison, we differentiated hESC to neural crest progenitors (NCP), a distinct peripheral nervous system progenitor population. Some epigenetic modifiers were regulated into the same direction in NEP and NCP, but also distinct differences were observed. For instance, the remodeling ATPase SMARCA2 was up-regulated >30-fold in NCP, while it remained unchanged in NEP; up-regulation of the ATP-dependent chromatin remodeler CHD7 was increased in NEP, while it was down-regulated in NCP. To compare the neural precursor profiles with those of mature neurons, we analyzed the epigenetic modifiers in human cortical tissue. This resulted in the identification of 30 regulations shared between all cell types, such as the histone methyltransferase SETD7. We also identified new markers for post-mitotic neurons, like the arginine methyl transferase PRMT8 and the methyl transferase EZH1. Our findings suggest a hitherto unexpected extent of regulation, and a cell type-dependent specificity of epigenetic modifiers in neurodifferentiation. PMID:22590590

  3. Genomic Aberrations Frequently Alter Chromatin Regulatory Genes in Chordoma

    PubMed Central

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F.; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C. David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-01-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ~8 Mb segment at 3p21.1–p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (~23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (~40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. PMID:27072194

  4. Alternative epigenetic chromatin states of polycomb target genes.

    PubMed

    Schwartz, Yuri B; Kahn, Tatyana G; Stenberg, Per; Ohno, Katsuhito; Bourgon, Richard; Pirrotta, Vincenzo

    2010-01-01

    Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development.

  5. Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin.

    PubMed Central

    Malik, N; Miwa, M; Sugimura, T; Thraves, P; Smulson, M

    1983-01-01

    Antibody to poly(ADP-ribose) has been covalently coupled to Sepharose and utilized to isolate selectively oligonucleosomes undergoing the poly(ADP-ribosyl)ation reaction from the bulk of chromatin. Approximately 12% of the unfractionated oligonucleosomes were bound to the immunoaffinity column and these represented essentially 100% of the original poly(ADP-ribosyl)ated nucleosomal species in the unfractionated chromatin. Poly(ADP-ribosyl)ated chromatin was not bound by preimmune IgG columns. KSCN eluted the modified nucleosomes in the form of nucleoprotein complexes. The eluted chromatin components were shown to contain poly(ADP-ribosyl)ated histones as well as automodified poly(ADP-ribose) polymerase. By using [3H]lysine- and [3H]arginine-labeled chromatin, it was shown that the poly-(ADP-ribosyl)ated histones, attached to stretches of oligonucleosomes bound to the column, had a 6-fold enrichment of the modification compared to histones of the unfractionated chromatin. This indicated that non-poly(ADP-ribosyl)ated nucleosomes, connected and proximal to the modified regions, were copurified by this procedure. This allowed characterization of the oligonucleosomal DNA around poly(ADP-ribosyl)ated chromatin domains to be compared with the unbound bulk chromatin. The data indicated that immunofractionated poly(ADP-ribosyl)ated oligonucleosomal DNA contained significant amounts of internal single-strand breaks compared with bulk chromatin. The bound nucleo-protein complexes were found to be enzymatically active for poly(ADP-ribose) polymerase after elution from the antibody column. In contrast, the unbound nucleosomes, representing 90% of the unfractionated chromatin, were totally inactive in the poly(ADP-ribosyl)ation reaction. Images PMID:6573670

  6. Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.

    PubMed

    Bailey, Aaron O; Panchenko, Tanya; Sathyan, Kizhakke M; Petkowski, Janusz J; Pai, Pei-Jing; Bai, Dina L; Russell, David H; Macara, Ian G; Shabanowitz, Jeffrey; Hunt, Donald F; Black, Ben E; Foltz, Daniel R

    2013-07-16

    Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.

  7. Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.

    PubMed

    Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina; Chagas da Cunha, Julia Pinheiro

    2017-01-01

    Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the

  8. Grating-flanked plasmonic coaxial apertures for efficient fiber optical tweezers.

    PubMed

    Saleh, Amr A E; Sheikhoelislami, Sassan; Gastelum, Steven; Dionne, Jennifer A

    2016-09-05

    Subwavelength plasmonic apertures have been foundational for direct optical manipulation of nanoscale specimens including sub-100 nm polymeric beads, metallic nanoparticles and proteins. While most plasmonic traps result in two-dimensional localization, three-dimensional manipulation has been demonstrated by integrating a plasmonic aperture on an optical fiber tip. However, such 3D traps are usually inefficient since the optical mode of the fiber and the subwavelength aperture only weakly couple. In this paper we design more efficient optical-fiber-based plasmonic tweezers combining a coaxial plasmonic aperture with a plasmonic grating coupler at the fiber tip facet. Using full-field finite difference time domain analysis, we optimize the grating design for both gold and silver fiber-based coaxial tweezers such that the optical transmission through the apertures is maximized. With the optimized grating, we show that the maximum transmission efficiency increases from 2.5% to 19.6% and from 1.48% to 16.7% for the gold and silver structures respectively. To evaluate their performance as optical tweezers, we calculate the optical forces and the corresponding trapping potential on dielectric particles interacting with the apertures. We demonstrate that the enahncement in the transmission translates into an equivalent increase in the optical forces. Consequently, the optical power required to achieve stable optical trapping is significantly reduced allowing for efficient localization and 3D manipulation of sub-30 nm dielectric particles.

  9. Composite Fiber Hazards

    DTIC Science & Technology

    1990-12-01

    34L boton Ion a tungsten boride core, and appear more like fine wires ,tin fibers. The fibers are combined with an epoxy matrix to form a prepreg j i...a 8-hour TWA Recommended Exposure Limit (REL) for fibrous glass of 3 fibers/cm3 for fibers with length >10 Jim and diameter ɛ.5 pm, and total

  10. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

    PubMed Central

    Sanborn, Adrian L.; Rao, Suhas S. P.; Huang, Su-Chen; Durand, Neva C.; Huntley, Miriam H.; Jewett, Andrew I.; Bochkov, Ivan D.; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher P.; Gnirke, Andreas; Melnikov, Alexandre; McKenna, Doug; Stamenova, Elena K.; Lander, Eric S.; Aiden, Erez Lieberman

    2015-01-01

    We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic “tension globule.” In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair. PMID:26499245

  11. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes.

    PubMed

    Sanborn, Adrian L; Rao, Suhas S P; Huang, Su-Chen; Durand, Neva C; Huntley, Miriam H; Jewett, Andrew I; Bochkov, Ivan D; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher P; Gnirke, Andreas; Melnikov, Alexandre; McKenna, Doug; Stamenova, Elena K; Lander, Eric S; Aiden, Erez Lieberman

    2015-11-24

    We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

  12. Fiber optic connector

    DOEpatents

    Rajic, Slobodan; Muhs, Jeffrey D.

    1996-01-01

    A fiber optic connector and method for connecting composite materials within which optical fibers are imbedded. The fiber optic connector includes a capillary tube for receiving optical fibers at opposing ends. The method involves inserting a first optical fiber into the capillary tube and imbedding the unit in the end of a softened composite material. The capillary tube is injected with a coupling medium which subsequently solidifies. The composite material is machined to a desired configuration. An external optical fiber is then inserted into the capillary tube after fluidizing the coupling medium, whereby the optical fibers are coupled.

  13. Fiber optic temperature sensor

    NASA Technical Reports Server (NTRS)

    Sawatari, Takeo (Inventor); Gaubis, Philip A. (Inventor)

    2000-01-01

    A fiber optic temperature sensor uses a light source which transmits light through an optical fiber to a sensor head at the opposite end of the optical fiber from the light source. The sensor head has a housing coupled to the end of the optical fiber. A metallic reflective surface is coupled to the housing adjacent the end of the optical fiber to form a gap having a predetermined length between the reflective surface and the optical fiber. A detection system is also coupled to the optical fiber which determines the temperature at the sensor head from an interference pattern of light which is reflected from the reflective surface.

  14. Fiber optic temperature sensor

    NASA Technical Reports Server (NTRS)

    Sawatari, Takeo (Inventor); Gaubis, Philip A. (Inventor); Mattes, Brenton L. (Inventor); Charnetski, Clark J. (Inventor)

    1999-01-01

    A fiber optic temperature sensor uses a light source which transmits light through an optical fiber to a sensor head at the opposite end of the optical fiber from the light source. The sensor head has a housing coupled to the end of the optical fiber. A metallic reflective surface is coupled to the housing adjacent the end of the optical fiber to form a gap having a predetermined length between the reflective surface and the optical fiber. A detection system is also coupled to the optical fiber which determines the temperature at the sensor head from an interference pattern of light which is reflected from the reflective surface.

  15. Fiber optic connector

    DOEpatents

    Rajic, S.; Muhs, J.D.

    1996-10-22

    A fiber optic connector and method for connecting composite materials within which optical fibers are imbedded are disclosed. The fiber optic connector includes a capillary tube for receiving optical fibers at opposing ends. The method involves inserting a first optical fiber into the capillary tube and imbedding the unit in the end of a softened composite material. The capillary tube is injected with a coupling medium which subsequently solidifies. The composite material is machined to a desired configuration. An external optical fiber is then inserted into the capillary tube after fluidizing the coupling medium, whereby the optical fibers are coupled. 3 figs.

  16. Coatings for graphite fibers

    NASA Technical Reports Server (NTRS)

    Galasso, F. S.; Scola, D. A.; Veltri, R. D.

    1980-01-01

    Graphite fibers released from composites during burning or an explosion caused shorting of electrical and electronic equipment. Silicon carbide, silica, silicon nitride and boron nitride were coated on graphite fibers to increase their electrical resistances. Resistances as high as three orders of magnitude higher than uncoated fiber were attained without any significant degradation of the substrate fiber. An organo-silicone approach to produce coated fibers with high electrical resistance was also used. Celion 6000 graphite fibers were coated with an organo-silicone compound, followed by hydrolysis and pyrolysis of the coating to a silica-like material. The shear and flexural strengths of composites made from high electrically resistant fibers were considerably lower than the shear and flexural strengths of composites made from the lower electrically resistant fibers. The lower shear strengths of the composites indicated that the coatings on these fibers were weaker than the coating on the fibers which were pyrolyzed at higher temperature.

  17. Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

    PubMed Central

    Poh, Huay Mei; Peh, Su Qin; Ong, Chin Thing; Zhang, Jingyao; Ruan, Xiaoan; Ruan, Yijun

    2012-01-01

    Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video. PMID:22564980

  18. Alumina fiber strength improvement

    NASA Technical Reports Server (NTRS)

    Pepper, R. T.; Nelson, D. C.

    1982-01-01

    The effective fiber strength of alumina fibers in an aluminum composite was increased to 173,000 psi. A high temperature heat treatment, combined with a glassy carbon surface coating, was used to prevent degradation and improve fiber tensile strength. Attempts to achieve chemical strengthening of the alumina fiber by chromium oxide and boron oxide coatings proved unsuccessful. A major problem encountered on the program was the low and inconsistent strength of the Dupont Fiber FP used for the investigation.

  19. Fiber optic monitoring device

    DOEpatents

    Samborsky, James K.

    1993-01-01

    A device for the purpose of monitoring light transmissions in optical fibers comprises a fiber optic tap that optically diverts a fraction of a transmitted optical signal without disrupting the integrity of the signal. The diverted signal is carried, preferably by the fiber optic tap, to a lens or lens system that disperses the light over a solid angle that facilitates viewing. The dispersed light indicates whether or not the monitored optical fiber or system of optical fibers is currently transmitting optical information.

  20. Photonic Crystal Fibers

    DTIC Science & Technology

    2005-12-01

    passive and active versions of each fiber designed under this task. Crystal Fibre shall provide characteristics of the fiber fabricated to include core...passive version of multicore fiber iteration 2. 15. SUBJECT TERMS EOARD, Laser physics, Fibre Lasers, Photonic Crystal, Multicore, Fiber Laser 16...9 00* 0 " CRYSTAL FIBRE INT ODUCTION This report describes the photonic crystal fibers developed under agreement No FA8655-o5-a- 3046. All