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Sample records for chromatin condensation modulates

  1. Efficient cell migration requires global chromatin condensation

    PubMed Central

    Gerlitz, Gabi; Bustin, Michael

    2010-01-01

    Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes. PMID:20530575

  2. Genome maintenance in the context of 4D chromatin condensation.

    PubMed

    Yu, Sonia; Yang, Fan; Shen, Wen H

    2016-08-01

    The eukaryotic genome is packaged in the three-dimensional nuclear space by forming loops, domains, and compartments in a hierarchical manner. However, when duplicated genomes prepare for segregation, mitotic cells eliminate topologically associating domains and abandon the compartmentalized structure. Alongside chromatin architecture reorganization during the transition from interphase to mitosis, cells halt most DNA-templated processes such as transcription and repair. The intrinsically condensed chromatin serves as a sophisticated signaling module subjected to selective relaxation for programmed genomic activities. To understand the elaborate genome-epigenome interplay during cell cycle progression, the steady three-dimensional genome requires a time scale to form a dynamic four-dimensional and a more comprehensive portrait. In this review, we will dissect the functions of critical chromatin architectural components in constructing and maintaining an orderly packaged chromatin environment. We will also highlight the importance of the spatially and temporally conscious orchestration of chromatin remodeling to ensure high-fidelity genetic transmission. PMID:27098512

  3. Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast

    PubMed Central

    Matsuda, Atsushi; Chikashige, Yuji; Ding, Da-Qiao; Ohtsuki, Chizuru; Mori, Chie; Asakawa, Haruhiko; Kimura, Hiroshi; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-01-01

    It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or ‘knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe. PMID:26205977

  4. 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. PMID:26459223

  5. Chromatin condensation of Xist genomic loci during oogenesis in mice

    PubMed Central

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2015-01-01

    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. PMID:26459223

  6. Class I Histone Deacetylase Thd1p Promotes Global Chromatin Condensation in Tetrahymena thermophila▿

    PubMed Central

    Parker, Kathryn; Maxson, Julia; Mooney, Alissa; Wiley, Emily A.

    2007-01-01

    Class I histone deacetylases (HDACs) regulate DNA-templated processes such as transcription. They act both at specific loci and more generally across global chromatin, contributing to acetylation patterns that may underlie large-scale chromatin dynamics. Although hypoacetylation is correlated with highly condensed chromatin, little is known about the contribution of individual HDACs to chromatin condensation mechanisms. Using the ciliated protozoan Tetrahymena thermophila, we investigated the role of a specific class I HDAC, Τhd1p, in the reversible condensation of global chromatin. In this system, the normal physiological response to cell starvation includes the widespread condensation of the macronuclear chromatin and general repression of gene transcription. We show that the chromatin in Thd1p-deficient cells failed to condense during starvation. The condensation failure correlated with aberrant hyperphosphorylation of histone H1 and the overexpression of CDC2, encoding the major histone H1 kinase. Changes in the rate of acetate turnover on core histones and in the distribution of acetylated lysines 9 and 23/27 on histone H3 isoforms that were found to correlate with normal chromatin condensation were absent from Thd1p mutant cells. These results point to a role for a class I HDAC in the formation of reversible higher-order chromatin structures and global genome compaction through mechanisms involving the regulation of H1 phosphorylation and core histone acetylation/deacetylation kinetics. PMID:17715364

  7. 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.

  8. 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. PMID:25870416

  9. Chromatin condensation in the erythrocytes of fish following exposure to cadmium

    SciTech Connect

    Gill, T.S.; Pant, J.C.

    1986-02-01

    Experimental cadmium poisoning in the fishes has been investigated and a variety of hematological changes have been observed. Studies on mammals have also revealed that Cd interferes with basic cellular processes and the metal has been shown to cause chromatin condensation and emptying of interchromatin spaces in cultured hepatocytes. The present work illustrates the effect of chronic Cd poisoning on the erythrocytes of a freshwater fish, Puntius conchonius with reference to chromatin condensation.

  10. 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

  11. 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

  12. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. PMID:27558729

  13. Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin

    PubMed Central

    Hiragami-Hamada, Kyoko; Soeroes, Szabolcs; Nikolov, Miroslav; Wilkins, Bryan; Kreuz, Sarah; Chen, Carol; De La Rosa-Velázquez, Inti A.; Zenn, Hans Michael; Kost, Nils; Pohl, Wiebke; Chernev, Aleksandar; Schwarzer, Dirk; Jenuwein, Thomas; Lorincz, Matthew; Zimmermann, Bastian; Walla, Peter Jomo; Neumann, Heinz; Baubec, Tuncay; Urlaub, Henning; Fischle, Wolfgang

    2016-01-01

    Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin. PMID:27090491

  14. Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin.

    PubMed

    Hiragami-Hamada, Kyoko; Soeroes, Szabolcs; Nikolov, Miroslav; Wilkins, Bryan; Kreuz, Sarah; Chen, Carol; De La Rosa-Velázquez, Inti A; Zenn, Hans Michael; Kost, Nils; Pohl, Wiebke; Chernev, Aleksandar; Schwarzer, Dirk; Jenuwein, Thomas; Lorincz, Matthew; Zimmermann, Bastian; Walla, Peter Jomo; Neumann, Heinz; Baubec, Tuncay; Urlaub, Henning; Fischle, Wolfgang

    2016-01-01

    Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin. PMID:27090491

  15. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

    PubMed

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-01-01

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies. PMID:27526631

  16. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

    PubMed Central

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-01-01

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies. PMID:27526631

  17. Chromatin Condensation and Enucleation in Red Blood Cells: An Open Question.

    PubMed

    Baron, Margaret H; Barminko, Jeffrey

    2016-03-01

    Differentiating erythroid cells undergo dramatic changes in morphology, with reduction in cell size, chromatin and nuclear condensation, and enucleation. In this issue of Developmental Cell, Zhao et al. (2016) show that these events are associated with the formation of transient, recurring nuclear openings and selective histone release mediated by caspase-3. PMID:26954541

  18. Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

    The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

  19. Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging.

    PubMed

    Spagnol, Stephen T; Dahl, Kris Noel

    2016-01-01

    The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

  20. Phosphorylation-Dependent Targeting of Tetrahymena HP1 to Condensed Chromatin

    PubMed Central

    Yale, Katerina; Tackett, Alan J.; Neuman, Monica; Bulley, Emily; Chait, Brian T.

    2016-01-01

    ABSTRACT The evolutionarily conserved proteins related to heterochromatin protein 1 (HP1), originally described in Drosophila, are well known for their roles in heterochromatin assembly and gene silencing. Targeting of HP1 proteins to specific chromatin locales is mediated, at least in part, by the HP1 chromodomain, which binds to histone H3 methylated at lysine 9 that marks condensed regions of the genome. Mechanisms that regulate HP1 targeting are emerging from studies with yeast and metazoans and point to roles for posttranslational modifications. Here, we report that modifications of an HP1 homolog (Hhp1) in the ciliate model Tetrahymena thermophila correlated with the physiological state and with nuclear differentiation events involving the restructuring of chromatin. Results support the model in which Hhp1 chromodomain binds lysine 27-methylated histone H3, and we show that colocalization with this histone mark depends on phosphorylation at a single Cdc2/Cdk1 kinase site in the “hinge region” adjacent to the chromodomain. These findings help elucidate important functional roles of reversible posttranslational modifications of proteins in the HP1 family, in this case, regulating the targeting of a ciliate HP1 to chromatin regions marked with methylated H3 lysine 27. IMPORTANCE Compacting the genome to various degrees influences processes that use DNA as a template, such as gene transcription and replication. This project was aimed at learning more about the cellular mechanisms that control genome compaction. Posttranslational modifications of proteins involved in genome condensation are emerging as potentially important points of regulation. To help elucidate protein modifications and how they affect the function of condensation proteins, we investigated the phosphorylation of the chromatin protein called Hhp1 in the ciliated protozoan Tetrahymena thermophila. This is one of the first functional investigations of these modifications of a nonhistone

  1. Phosphorylation-Dependent Targeting of Tetrahymena HP1 to Condensed Chromatin.

    PubMed

    Yale, Katerina; Tackett, Alan J; Neuman, Monica; Bulley, Emily; Chait, Brian T; Wiley, Emily

    2016-01-01

    The evolutionarily conserved proteins related to heterochromatin protein 1 (HP1), originally described in Drosophila, are well known for their roles in heterochromatin assembly and gene silencing. Targeting of HP1 proteins to specific chromatin locales is mediated, at least in part, by the HP1 chromodomain, which binds to histone H3 methylated at lysine 9 that marks condensed regions of the genome. Mechanisms that regulate HP1 targeting are emerging from studies with yeast and metazoans and point to roles for posttranslational modifications. Here, we report that modifications of an HP1 homolog (Hhp1) in the ciliate model Tetrahymena thermophila correlated with the physiological state and with nuclear differentiation events involving the restructuring of chromatin. Results support the model in which Hhp1 chromodomain binds lysine 27-methylated histone H3, and we show that colocalization with this histone mark depends on phosphorylation at a single Cdc2/Cdk1 kinase site in the "hinge region" adjacent to the chromodomain. These findings help elucidate important functional roles of reversible posttranslational modifications of proteins in the HP1 family, in this case, regulating the targeting of a ciliate HP1 to chromatin regions marked with methylated H3 lysine 27. IMPORTANCE Compacting the genome to various degrees influences processes that use DNA as a template, such as gene transcription and replication. This project was aimed at learning more about the cellular mechanisms that control genome compaction. Posttranslational modifications of proteins involved in genome condensation are emerging as potentially important points of regulation. To help elucidate protein modifications and how they affect the function of condensation proteins, we investigated the phosphorylation of the chromatin protein called Hhp1 in the ciliated protozoan Tetrahymena thermophila. This is one of the first functional investigations of these modifications of a nonhistone chromatin

  2. Chromatin-modifying enzymes as modulators of reprogramming.

    PubMed

    Onder, Tamer T; Kara, Nergis; Cherry, Anne; Sinha, Amit U; Zhu, Nan; Bernt, Kathrin M; Cahan, Patrick; Marcarci, B Ogan; Unternaehrer, Juli; Gupta, Piyush B; Lander, Eric S; Armstrong, Scott A; Daley, George Q

    2012-03-29

    Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. PMID:22388813

  3. The Dynamics of HCF-1 Modulation of Herpes Simplex Virus Chromatin during Initiation of Infection

    PubMed Central

    Vogel, Jodi L.; Kristie, Thomas M.

    2013-01-01

    Successful infection of herpes simplex virus is dependent upon chromatin modulation by the cellular coactivator host cell factor-1 (HCF-1). This review focuses on the multiple chromatin modulation components associated with HCF-1 and the chromatin-related dynamics mediated by this coactivator that lead to the initiation of herpes simplex virus (HSV) immediate early gene expression. PMID:23698399

  4. Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

    SciTech Connect

    Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan; Koury, Mark J.; Ney, Paul A.; Chasis, Joel Anne; Grigoryev, Sergei A.

    2008-08-21

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

  5. Linker histones are not essential and affect chromatin condensation in vivo.

    PubMed

    Shen, X; Yu, L; Weir, J W; Gorovsky, M A

    1995-07-14

    We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila. These disruptions are shown to eliminate completely the expression of each protein. Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival. Histone H1 knockout (delta H1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (delta MicLH) cells have enlarged micronuclei and normal-sized macronuclei. delta MicLH cells undergo mitosis normally. However, the micronuclear mitotic chromosome structure is less condensed. These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo. PMID:7606784

  6. Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

    PubMed Central

    Talebi, Ali Reza; Khalili, Mohammad Ali; Vahidi, Serajodin; Ghasemzadeh, Jalal; Tabibnejad, Nasim

    2013-01-01

    Objectives To evaluate the effect of cord injury on (1) sperm parameters and (2) DNA chromatin status. Design Case–control study. Setting Data were collected from men referred to Research and Clinical Center for Infertility, Yazd, Iran. Participants Thirty infertile men with the presence of any level of spinal cord injury (SCI) were compared with 30 healthy donors with definite fertility and normal sperm parameters. Interventions Not applicable. Outcome measures Sperm chromatin integrity was assessed using aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. The rate of apoptotic spermatozoa was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results Sperm concentration, motility, and morphology in men with SCI were significantly decreased compared with control group (P < 0.05). In addition, with regard to cytochemical staining and TUNEL test, the rate of reacted spermatozoa was increased significantly in SCI group when compared with the controls (P < 0.05). The majority of AB, TB, AO, and CMA3-reacted spermatozoa were higher than the “cut-off” value in men with SCI, as were the number of apoptotic spermatozoa stained with TUNEL. Conclusion Results showed that SCI disturbs sperm parameters, nuclear maturity, and DNA integrity of spermatozoa. Therefore, the production of spermatozoa with less condensed chromatin and more apoptotic rate increases after cord injury and this may be one possible cause of infertility following SCI. PMID:23809529

  7. EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells Against Ionizing Radiation

    PubMed Central

    Wang, Meng; Kern, Ashley M.; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H.; Efstathiou, Jason A.; Settleman, Jeff; Willers, Henning

    2014-01-01

    Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in-vitro and in-vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like co-localization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is co-dependent upon EGFR and PKCα. PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. PMID:24648348

  8. A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis[W][OPEN

    PubMed Central

    Waidmann, Sascha; Kusenda, Branislav; Mayerhofer, Juliane; Mechtler, Karl; Jonak, Claudia

    2014-01-01

    Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function. PMID:25387881

  9. 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

  10. 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

  11. Chromatin condensation, cysteine-rich protamine, and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda).

    PubMed

    Gimenez-Bonafé, Pepita; Ribes, Enric; Sautière, Pierre; Gonzalez, Angel; Kasinsky, Harold; Kouach, Mustafa; Sautière, Pierre-Eric; Ausió, Juan; Chiva, Manel

    2002-06-01

    During spermiogenesis in Eledone cirrhosa a single protamine substitutes for histones in nuclei of developing spermatids. This protein displays a peculiar primary structure. It contains 22.6 mol% cysteine residues (19 cysteines in 84 residues). This makes it the most cysteine-rich protamine known. The proportion of basic residues is relatively low (arginine 36.9 mol%, lysine 19.0 mol%). The protamine of E. cirrhosa condenses spermiogenic chromatin in a pattern which comprises fibres with a progressively larger diameter and lamellae that finally undergo definitive coalescence. We have also performed a study that estimates the number of interprotamine disulphide bonds formed during the process of spermiogenic chromatin condensation by means of sequential disappearance of MMNA (monomaleimido-nanogold) labelling. During the first step of spermiogenesis, protamines are found spread over very slightly condensed chromatin with their cysteines in a reactive state (protamine-cys-SH). From this stage the interprotamine disulphide bonds are established in a progressive way. First they are formed inside the chromatin fibres. Subsequently, they participate in the mechanism of fibre coalescence and finally, in the last step of spermiogenesis, the remaining free reactive -SH groups of cysteine form disulphide bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus. PMID:12113475

  12. Dynamic modulation of HSV chromatin drives initiation of infection and provides targets for epigenetic therapies

    PubMed Central

    Kristie, Thomas M.

    2015-01-01

    Upon infection, the genomes of herpesviruses undergo a striking transition from a non-nucleosomal structure to a chromatin structure. The rapid assembly and modulation of nucleosomes during the initial stage of infection results in an overlay of complex regulation that requires interactions of a plethora of chromatin modulation components. For herpes simplex virus, the initial chromatin dynamic is dependent on viral and host cell transcription factors and coactivators that mediate the balance between heterochromatic suppression of the viral genome and the euchromatin transition that allows and promotes the expression of viral immediate early genes. Strikingly similar to lytic infection, in sensory neurons this dynamic transition between heterochromatin and euchromatin governs the establishment, maintenance, and reactivation from the latent state. Chromatin dynamics in both the lytic infection and latency-reactivation cycles provides opportunities to shift the balance using small molecule epigenetic modulators to suppress viral infection, shedding, and reactivation from latency. PMID:25702087

  13. 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

  14. 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.

  15. An Essential Viral Transcription Activator Modulates Chromatin Dynamics

    PubMed Central

    Gibeault, Rebecca L.; Bildersheim, Michael D.

    2016-01-01

    Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes. PMID:27575707

  16. Effect of different thawing temperatures on the viability, in vitro fertilizing capacity and chromatin condensation of frozen boar semen packaged in 5 ml straws.

    PubMed

    Córdova-Izquierdo, A; Oliva, J H; Lleó, B; García-Artiga, C; Corcuera, B D; Pérez-Gutiérrez, J F

    2006-03-01

    The effect of two different thawing temperatures on frozen boar semen viability, in vitro fertilizing capacity and chromatin condensation and stability was studied. Freeze-thaw motility, normal apical ridge (NAR), in vitro fertilizing (IVF) capacity and chromatin condensation and stability were evaluated after thawing at 42 degrees C, 40s and 50 degrees C, 40s. Chromatin condensation degree was determined by flow cytometry, using propidium iodide as fluorochrome intercalating agent, and chromatin stability was evaluated by the same procedure after inducing sperm chromatin decondensation with ethylene diamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS). The results showed that thawing straws at 42 degrees C, 40s significantly reduced motility compared to straws thawed at 50 degrees C, 40s. NAR, penetration, monospermy and polyspermy were not different between the two groups of samples thawed at different temperatures. Chromatin was significantly more compact when thawing was performed at 50 degrees C, but its stability did not show any difference relative to thawing at 42 degrees C. It is suggested that the interactions involved in chromatin overcondensation had a non-covalent nature. PMID:15975744

  17. Etoposide interferes with the process of chromatin condensation during alga Chara vulgaris spermiogenesis.

    PubMed

    Agnieszka, Wojtczak

    2014-10-01

    DNA topoisomerase II plays an essential role in animal spermiogenesis, where changes of chromatin structure are connected with appearance of transient DNA breaks. Such topo II activity can be curtailed by inhibitors such as etoposide and suramine. The aim of the present study was to investigate, for the first time, the effect of etoposide on spermatid chromatin remodeling in the green alga Chara vulgaris. This inhibitor prolonged the early spermiogenesis stages and blocked the formation of the phosphorylated form of histone H2AX at stages VI-VII. The lack of transient DSBs at these stages impairs the elimination of supercoils containing nucleosomes which lead to disturbances in nucleoprotein exchange and the pattern of spermatid chromatin fibrils at stages VI-VIII. Immunofluorescent and ultrastructural observations revealed that during C. vulgaris spermiogenesis topo II played an important role similar to that in mammals. Some corresponding features had been pointed out before, the present studies showed further similarities. PMID:25041830

  18. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation.

    PubMed

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs' function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR's combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  19. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation

    PubMed Central

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs’ function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR’s combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  20. Dynamics of histone H2A, H4 and HS1ph during spermatogenesis with a focus on chromatin condensation and maturity of spermatozoa

    PubMed Central

    Zhang, Zhao-Hui; Mu, Shu-Mei; Guo, Ming-Shen; Wu, Jiang-li; Li, Yan-qin; Zhang, Han; Wang, Ying; Kang, Xian-Jiang

    2016-01-01

    Histones and histone phosphorylation play vital roles during animal spermatogenesis and spermatozoa maturation. The dynamic distribution of histones H2A and H4 and phosphorylated H2A and H4 at serine 1 (HS1ph) was explored in mammalian and Decapoda germ cells, with a special focus on the distribution of H2A, H4 and HS1ph between mouse condensed spermatozoa chromatin and crab non-condensed spermatozoa chromatin. The distribution of histone marks was also analysed in mature spermatozoa with different chromatin structures. Histone H2A and H4 marks were closely associated with the relatively loose chromatin structure in crab spermatozoa. The significant decrease in the HS1ph signal during spermatogenesis suggests that eliminating most of these epigenetic marks in the nucleusis closely associated with spermatozoa maturity. PMID:27121047

  1. Piwi Modulates Chromatin Accessibility by Regulating Multiple Factors Including Histone H1 to Repress Transposons.

    PubMed

    Iwasaki, Yuka W; Murano, Kensaku; Ishizu, Hirotsugu; Shibuya, Aoi; Iyoda, Yumiko; Siomi, Mikiko C; Siomi, Haruhiko; Saito, Kuniaki

    2016-08-01

    PIWI-interacting RNAs (piRNAs) mediate transcriptional and post-transcriptional silencing of transposable element (TE) in animal gonads. In Drosophila ovaries, Piwi-piRNA complexes (Piwi-piRISCs) repress TE transcription by modifying the chromatin state, such as by H3K9 trimethylation. Here, we demonstrate that Piwi physically interacts with linker histone H1. Depletion of Piwi decreases H1 density at a subset of TEs, leading to their derepression. Silencing at these loci separately requires H1 and H3K9me3 and heterochromatin protein 1a (HP1a). Loss of H1 increases target loci chromatin accessibility without affecting H3K9me3 density at these loci, while loss of HP1a does not impact H1 density. Thus, Piwi-piRISCs require both H1 and HP1a to repress TEs, and the silencing is correlated with the chromatin state rather than H3K9me3 marks. These findings suggest that Piwi-piRISCs regulate the interaction of chromatin components with target loci to maintain silencing of TEs through the modulation of chromatin accessibility. PMID:27425411

  2. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers

    PubMed Central

    Ruthenburg, Alexander J; Allis, C David; Patel, Dinshaw J

    2015-01-01

    Histones comprise the major protein component of chromatin, the scaffold in which the eukaryotic genome is packaged, and are subject to many types of post-translational modifications (PTMs), especially on their flexible tails. These modifications may constitute a ‘histone code’ and could be used to manage epigenetic information that helps extend the genetic message beyond DNA sequences. This proposed code, read in part by histone PTM–binding ‘effector’ modules and their associated complexes, is predicted to define unique functional states of chromatin and/or regulate various chromatin-templated processes. A wealth of structural and functional data show how chromatin effector modules target their cognate covalent histone modifications. Here we summarize key features in molecular recognition of histone PTMs by a diverse family of ‘reader pockets’, highlighting specific readout mechanisms for individual marks, common themes and insights into the downstream functional consequences of the interactions. Changes in these interactions may have far-reaching implications for human biology and disease, notably cancer. PMID:17984965

  3. Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications.

    PubMed

    Kristie, Thomas M

    2016-01-01

    Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes. PMID:26884430

  4. Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations

    PubMed Central

    Visvanathan, Ashwat; Ahmed, Kashif; Even-Faitelson, Liron; Lleres, David; Bazett-Jones, David P.; Lamond, Angus I.

    2013-01-01

    The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism. PMID:23840764

  5. Packaging, Transportation and Recycling of NPP Condenser Modules - 12262

    SciTech Connect

    Polley, G.M.

    2012-07-01

    Perma-Fix was awarded contract from Energy Northwest for the packaging, transportation and disposition of the condenser modules, water boxes and miscellaneous metal, combustibles and water generated during the 2011 condenser replacement outage at the Columbia Generating Station. The work scope was to package the water boxes and condenser modules as they were removed from the facility and transfer them to the Perma-Fix Northwest facility for processing, recycle of metals and disposition. The condenser components were oversized and overweight (the condenser modules weighed ∼102,058 kg [225,000 lb]) which required special equipment for loading and transport. Additional debris waste was packaged in inter-modals and IP-1 boxes for transport. A waste management plan was developed to minimize the generation of virtually any waste requiring landfill disposal. The Perma-Fix Northwest facility was modified to accommodate the ∼15 m [50-ft] long condenser modules and equipment was designed and manufactured to complete the disassembly, decontamination and release survey. The condenser modules are currently undergoing processing for free release to a local metal recycler. Over three millions pounds of metal will be recycled and over 95% of the waste generated during this outage will not require land disposal. There were several elements of this project that needed to be addressed during the preparation for this outage and the subsequent packaging, transportation and processing. - Staffing the project to support 24/7 generation of large components and other wastes. - The design and manufacture of the soft-sided shipping containers for the condenser modules that measured ∼15 m X 4 m X 3 m [50 ft X 13 ft X 10 ft] and weighed ∼102,058 kg [225,000 lbs] - Developing a methodology for loading the modules into the shipping containers. - Obtaining a transport vehicle for the modules. - Designing and modifying the processing facility. - Movement of the modules at the processing

  6. The CW domain, a new histone recognition module in chromatin proteins.

    PubMed

    Hoppmann, Verena; Thorstensen, Tage; Kristiansen, Per Eugen; Veiseth, Silje Veie; Rahman, Mohummad Aminur; Finne, Kenneth; Aalen, Reidunn B; Aasland, Rein

    2011-05-18

    Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states. PMID:21522130

  7. Modulation of chromatin position and gene expression by HDAC4 interaction with nucleoporins

    PubMed Central

    Kehat, Izhak; Accornero, Federica; Aronow, Bruce J.

    2011-01-01

    Class IIa histone deacetylases (HDACs) can modulate chromatin architecture and transcriptional activity, thereby participating in the regulation of cellular responses such as cardiomyocyte hypertrophy. However, the target genes of class IIa HDACs that control inducible cardiac growth and the broader mechanisms whereby these deacetylases modulate locus-specific gene expression within chromatin remain a mystery. Here, we used genome-wide promoter occupancy analysis, expression profiling, and primary cell validation to identify direct class IIa HDAC4 targets in cardiomyocytes. Simultaneously, we identified nucleoporin155 (Nup155) as an HDAC4-interacting protein. Mechanistically, we show that HDAC4 modulated the association of identified target genes with nucleoporins through interaction with Nup155. Moreover, a truncated mutant of Nup155 that cannot bind HDAC4 suppressed HDAC4-induced gene expression patterns and chromatin–nucleoporin association, suggesting that Nup155-mediated localization was required for HDAC4’s effect on gene expression. We thus propose a novel mechanism of action for HDAC4, suggesting it can function to dynamically regulate gene expression through changes in chromatin–nucleoporin association. PMID:21464227

  8. 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. PMID:25262620

  9. Downregulation of SWI/SNF chromatin remodeling factor subunits modulates cisplatin cytotoxicity

    SciTech Connect

    Kothandapani, Anbarasi; Gopalakrishnan, Kathirvel; Kahali, Bhaskar; Reisman, David; Patrick, Steve M.

    2012-10-01

    Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions. -- Highlights: Black-Right-Pointing-Pointer Stable knockdown of Brg1 and Brm enhances cellular sensitivity to cisplatin. Black-Right-Pointing-Pointer Downregulation of Brg1 and Brm impedes the repair of cisplatin intrastrand adducts and interstrand crosslinks. Black-Right-Pointing-Pointer Brg1 and Brm deficiency results in impaired chromatin relaxation, altered checkpoint activation as well as enhanced apoptosis. Black-Right-Pointing-Pointer Downregulation of Brg1 and Brm affects recruitment of ERCC1, but not XPC to cisplatin DNA lesions.

  10. Structure and Function of the SWIRM Domain, a Conserved Protein Module Found in Chromatin Regulatory Complexes

    SciTech Connect

    Da,G.; Lenkart, J.; Zhao, K.; Shiekhattar, R.; Cairns, B.; Marmorstein, R.

    2006-01-01

    The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the in vitro and in vivo function of the SWIRM domains from Saccharomyces cerevisiae Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix-turn-helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA-binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.

  11. PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC).

    PubMed

    Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei; Zhu, Jiang; Ozaki, Toshinori; Bu, Youquan

    2015-03-13

    Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent with those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. PMID:25666944

  12. Hydrogen sulfide attenuates cytokine production through the modulation of chromatin remodeling

    PubMed Central

    RIOS, ESTER C.S.; SZCZESNY, BARTOSZ; SORIANO, FRANCISCO G.; OLAH, GABOR; SZABO, CSABA

    2015-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 μg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors. PMID:25873160

  13. PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC)

    SciTech Connect

    Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei; Zhu, Jiang; Ozaki, Toshinori; Bu, Youquan

    2015-03-13

    Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent with those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. - Highlights: • PRR11 started to increase in the late S phase and was retained until just before mitotic telophase. • PRR11-knockdown caused a significant cell cycle arrest in the late S phase and G2 phase. • The treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. • PRR11-knockdown led to multipolar spindles and multiple nuclei. • Forced expression of PRR11 promoted the PCC and inhibited

  14. Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

    PubMed Central

    Gertych, Arkadiusz; Tajbakhsh, Jian

    2013-01-01

    This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns — visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis — in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors. PMID:23562889

  15. Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

    SciTech Connect

    Komura, Jun-ichiro; Ikehata, Hironobu; Mori, Toshio; Ono, Tetsuya

    2012-03-10

    During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.

  16. Transcriptional Control by PARP-1: Chromatin Modulation, Enhancer-binding, Coregulation, and Insulation

    PubMed Central

    Kraus, W. Lee

    2008-01-01

    Summary The regulation of gene expression requires a wide array of protein factors that can modulate chromatin structure, act at enhancers, function as transcriptional coregulators, or regulate insulator function. Poly(ADP-ribose) polymerase-1 (PARP-1), an abundant and ubiquitous nuclear enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been implicated in all of these functions. Recent biochemical, genomic, proteomic, and cell-based studies have highlighted the role of PARP-1 in each of these processes and provided new insights about the molecular mechanisms governing PARP-1-dependent regulation of gene expression. In addition, these studies have demonstrated how PARP-1 functions as an integral part of cellular signaling pathways that culminate in gene regulatory outcomes. PMID:18450439

  17. Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

    PubMed Central

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; Martin, Eric W.; Krietsch, Jana; Sanchez, Gabriela; Inman, Margaux; Tsang, Herman; Warr, Matthew; Passegué, Emmanuelle; Larabell, Carolyn A.; Forsberg, E. Camilla

    2015-01-01

    Summary Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation. PMID:26489895

  18. The role of chromatin structure in cell migration

    PubMed Central

    Gerlitz, Gabi; Bustin, Michael

    2010-01-01

    Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization seems to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow cellular passage through narrow openings, is dependent not only on changes in cytoskeletal elements, but also on the global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure may facilitate cellular reorganizations necessary for efficient migration. PMID:20951589

  19. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

    PubMed

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  20. A phylogenetic study of SPBP and RAI1: evolutionary conservation of chromatin binding modules.

    PubMed

    Darvekar, Sagar; Rekdal, Cecilie; Johansen, Terje; Sjøttem, Eva

    2013-01-01

    Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or "reader" modules. The nuclear proteins SPBP and RAI1 are composed of several putative "reader" modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation. PMID:24205348

  1. A Phylogenetic Study of SPBP and RAI1: Evolutionary Conservation of Chromatin Binding Modules

    PubMed Central

    Darvekar, Sagar; Rekdal, Cecilie; Johansen, Terje; Sjøttem, Eva

    2013-01-01

    Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or “reader” modules. The nuclear proteins SPBP and RAI1 are composed of several putative “reader” modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation. PMID:24205348

  2. Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape.

    PubMed

    Camp, J Gray; Frank, Christopher L; Lickwar, Colin R; Guturu, Harendra; Rube, Tomas; Wenger, Aaron M; Chen, Jenny; Bejerano, Gill; Crawford, Gregory E; Rawls, John F

    2014-09-01

    Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs. PMID:24963153

  3. Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape

    PubMed Central

    Camp, J. Gray; Frank, Christopher L.; Lickwar, Colin R.; Guturu, Harendra; Rube, Tomas; Wenger, Aaron M.; Chen, Jenny; Bejerano, Gill; Crawford, Gregory E.

    2014-01-01

    Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs. PMID:24963153

  4. Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation

    SciTech Connect

    Mannioui, Abdelkrim . E-mail: karim.mannioui@chu-stlouis.fr; Schiffer, Cecile . E-mail: cecile.schiffer@voila.fr; Felix, Nathalie . E-mail: nathalie.felix@chu-stlouis.fr

    2004-11-10

    We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.

  5. Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions.

    PubMed

    Sasaki, Narie; Kuroiwa, Haruko; Nishitani, Chikako; Takano, Hiroyoshi; Higashiyama, Tetsuya; Kobayashi, Tamaki; Shirai, Yuki; Sakai, Atsushi; Kawano, Shigeyuki; Murakami-Murofushi, Kimiko; Kuroiwa, Tsuneyoshi

    2003-12-01

    Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions. PMID:12960433

  6. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription

    PubMed Central

    Ramakrishnan, Saravanan; Pokhrel, Srijana; Palani, Sowmiya; Pflueger, Christian; Parnell, Timothy J.; Cairns, Bradley R.; Bhaskara, Srividya; Chandrasekharan, Mahesh B.

    2016-01-01

    Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation−demethylation together control chromatin dynamics during various facets of transcriptional regulation. PMID:27325136

  7. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription.

    PubMed

    Ramakrishnan, Saravanan; Pokhrel, Srijana; Palani, Sowmiya; Pflueger, Christian; Parnell, Timothy J; Cairns, Bradley R; Bhaskara, Srividya; Chandrasekharan, Mahesh B

    2016-01-01

    Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation-demethylation together control chromatin dynamics during various facets of transcriptional regulation. PMID:27325136

  8. Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive.

    PubMed

    Gatchalian, Jovylyn; Gallardo, Carmen Mora; Shinsky, Stephen A; Ospina, Ruben Rosas; Liendo, Andrea Mansilla; Krajewski, Krzysztof; Klein, Brianna J; Andrews, Forest H; Strahl, Brian D; M van Wely, Karel H; Kutateladze, Tatiana G

    2016-07-27

    Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division. PMID:27016734

  9. GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

    PubMed

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development. PMID:17984219

  10. Modulating chromatin structure and DNA accessibility by deacetylase inhibition enhances the anti-cancer activity of silver nanoparticles.

    PubMed

    Igaz, Nóra; Kovács, Dávid; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M; Kiricsi, Mónika

    2016-10-01

    Histone deacetylase (HDAC) inhibitors are considered as novel therapeutic agents inducing cell cycle arrest and apoptotic cell death in various cancer cells. Inhibition of deacetylase activity results in a relaxed chromatin structure thereby rendering the genetic material more vulnerable to DNA targeting agents that could be exploited by combinational cancer therapy. The unique potential of silver nanoparticles (AgNPs) in tumor therapy relies on the generation of reactive radicals which trigger oxidative stress, DNA damage and apoptosis in cancer cells. The revolutionary application of AgNPs as chemotherapeutical drugs seems very promising, nevertheless the exact molecular mechanisms of AgNP action in combination with other anti-cancer agents have yet to be elucidated in details before clinical administrations. As a step towards this we investigated the combinational effect of HDAC inhibition and AgNP administration in HeLa cervical cancer cells. We identified synergistic inhibition of cancer cell growth and migration upon combinational treatments. Here we report that the HDAC inhibitor Trichostatin A enhances the DNA targeting capacity and apoptosis inducing efficacy of AgNPs most probably due to its effect on chromatin condensation. These results point to the potential benefits of combinational application of HDAC inhibitors and AgNPs in novel cancer medication protocols. PMID:27434153

  11. MIDGET Unravels Functions of the Arabidopsis Topoisomerase VI Complex in DNA Endoreduplication, Chromatin Condensation, and Transcriptional Silencing[W

    PubMed Central

    Kirik, Viktor; Schrader, Andrea; Uhrig, Joachim F.; Hulskamp, Martin

    2007-01-01

    The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles. PMID:17951446

  12. LincRNA-Cox2 Promotes Late Inflammatory Gene Transcription in Macrophages through Modulating SWI/SNF-Mediated Chromatin Remodeling.

    PubMed

    Hu, Guoku; Gong, Ai-Yu; Wang, Yang; Ma, Shibin; Chen, Xiqiang; Chen, Jing; Su, Chun-Jen; Shibata, Annemarie; Strauss-Soukup, Juliane K; Drescher, Kristen M; Chen, Xian-Ming

    2016-03-15

    Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which was recently shown to mediate the activation and repression of distinct classes of immune genes in innate immune cells. We report that lincRNA-Cox2, located at chromosome 1 proximal to the PG-endoperoxide synthase 2 (Ptgs2/Cox2) gene, is an early-primary inflammatory gene controlled by NF-κB signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-κB-regulated late-primary inflammatory response genes stimulated by bacterial LPS. Specifically, lincRNA-Cox2 is assembled into the switch/sucrose nonfermentable (SWI/SNF) complex in cells after LPS stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-κB subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory-response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role for NF-κB-induced lincRNA-Cox2 as a coactivator of NF-κB for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling. PMID:26880762

  13. Removal of excitations of Bose-Einstein condensates by space- and time-modulated potentials

    SciTech Connect

    Staliunas, Kestutis

    2011-07-15

    We propose that periodically in space- and time-modulated potentials (dynamic lattices) can efficiently remove the excited (the high-energy and large momentum) components of the trapped Bose-Einstein condensates (BECs) and, consequently, can result in efficient cleaning of the BECs. We prove the idea by numerically solving the mean-field models (the Schroedinger equation for noninteracting condensates and the Gross-Pitaevskii equation for interacting condensates of repulsive atoms), and we evaluate parameters and conditions for the efficient removal of excitations.

  14. Infrasound induced instability by modulation of condensation process in the atmosphere.

    PubMed

    Naugolnykh, Konstantin; Rybak, Samuil

    2008-12-01

    A sound wave in supersaturated water vapor can modulate both the process of heat release caused by condensation, and subsequently, as a result, the resonance interaction of sound with the modulated heat release provides sound amplification. High-intensity atmospheric perturbations such as cyclones and thunderstorms generate infrasound, which is detectable at large distances from the source. The wave-condensation instability can lead to variation in the level of infrasound radiation by a developing cyclone, and this can be as a precursor of these intense atmospheric events. PMID:19206769

  15. Full-time dynamics of modulational instability in spinor Bose-Einstein condensates

    SciTech Connect

    Doktorov, Evgeny V.; Rothos, Vassilis M.; Kivshar, Yuri S.

    2007-07-15

    We describe the full-time dynamics of modulational instability in F=1 spinor Bose-Einstein condensates for the case of the integrable three-component model associated with the matrix nonlinear Schroedinger equation. We obtain an exact homoclinic solution of this model by employing the dressing method which we generalize to the case of the higher-rank projectors. This homoclinic solution describes the development of modulational instability beyond the linear regime, and we show that the modulational instability demonstrates the reversal property when the growth of the modulated amplitude is changed by its exponential decay.

  16. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.

    PubMed

    Cain, K; Inayat-Hussain, S H; Couet, C; Qin, H M; Oberhammer, F A

    1996-04-01

    This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1. PMID:8900474

  17. Bose-Einstein condensates under a spatially modulated transverse confinement

    SciTech Connect

    Salasnich, L.; Toigo, F.; Cetoli, A.; Malomed, B. A.; Reatto, L.

    2007-07-15

    We derive an effective nonpolynomial Schroedinger equation (NPSE) for self-repulsive or attractive BEC in the nearly one-dimensional cigar-shaped trap, with the transverse confining frequency periodically modulated along the axial direction. In addition to the usual linear cigar-shaped trap, where the periodic modulation emulates the action of an optical lattice (OL), the model may be also relevant to toroidal traps, where an ordinary OL cannot be created. For either sign of the nonlinearity, extended and localized states are found, in the numerical form [using both the effective NPSE and the full three-dimensional (3D) Gross-Pitaevskii equation] and by means of the variational approximation (VA). The latter is applied to construct ground-state solitons and predict the collapse threshold in the case of self-attraction. It is shown that numerical solutions provided by the one-dimensional NPSE are always very close to full 3D solutions, and the VA yields quite reasonable results too. The transition from delocalized states to gap solitons, in the first finite bandgap of the linear spectrum, is examined in detail, for the repulsive and attractive nonlinearities alike.

  18. Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus.

    PubMed

    Miller, Kelly E; Heald, Rebecca

    2015-04-27

    Linker histone H1 is required for mitotic chromosome architecture in Xenopus laevis egg extracts and, unlike core histones, exhibits rapid turnover on chromatin. Mechanisms regulating the recruitment, deposition, and dynamics of linker histones in mitosis are largely unknown. We found that the cytoplasmic histone chaperone nucleosome assembly protein 1 (Nap1) associates with the embryonic isoform of linker histone H1 (H1M) in egg extracts. Immunodepletion of Nap1 decreased H1M binding to mitotic chromosomes by nearly 50%, reduced H1M dynamics as measured by fluorescence recovery after photobleaching and caused chromosome decondensation similar to the effects of H1M depletion. Defects in H1M dynamics and chromosome condensation were rescued by adding back wild-type Nap1 but not a mutant lacking sites subject to posttranslational modification by glutamylation. Nap1 glutamylation increased the deposition of H1M on sperm nuclei and chromatin-coated beads, indicating that charge-shifting posttranslational modification of Nap1 contributes to H1M dynamics that are essential for higher order chromosome architecture. PMID:25897082

  19. Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.

    PubMed

    Rybaczek, Dorota; Maszewski, Janusz

    2007-01-01

    Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA. PMID:17111099

  20. A Kinetic Approach to Bose-Einstein Condensates: Self-Phase Modulation and Bogoliubov Oscillations

    SciTech Connect

    Mendonca, J.T.; Bingham, R.; Shukla, P.K.

    2005-11-01

    A kinetic approach to Bose-Einstein condensates (BECs) is proposed based on the Wigner-Moyal equation (WME). In the semiclassical limit, the WME reduces to the particle-number conservation equation. Two examples of applications are (i) a self-phase modulation of a BE condensate beam, where we show that part of the beam is decelerated and eventually stops as a result of the gradient of the effective self-potential, and (ii) the derivation of a kinetic dispersion relation for sound waves in BECs, including collisionless Landau damping.

  1. The p23 molecular chaperone and GCN5 acetylase jointly modulate protein-DNA dynamics and open chromatin status

    PubMed Central

    Zelin, Elena; Zhang, Yang; Toogun, Oyetunji A.; Zhong, Sheng; Freeman, Brian C.

    2012-01-01

    SUMMARY Cellular processes function through multi-step pathways that are reliant on the controlled association and disassociation of sequential protein complexes. While dynamic action is critical to propagate and terminate work, the mechanisms used to disassemble biological structures are not fully understood. Here, we show that the p23 molecular chaperone initiates disassembly of protein-DNA complexes and that the GCN5 acetyltransferase prolongs the dissociated state through lysine acetylation. By modulating the DNA bound-state, we found that the conserved and essential joint activities of p23 and GCN5 impacted transcription factor activation potential and response time to an environmental cue. Notably, p23 and GCN5 were required to maintain open chromatin regions along the genome indicating that dynamic protein behavior is a critical feature of various DNA-associated events. Our data support a model in which p23 and GCN5 regulate diverse multi-step pathways by controlling the longevity of protein-DNA complexes. PMID:23022381

  2. Facilitation of base excision repair by chromatin remodeling.

    PubMed

    Hinz, John M; Czaja, Wioletta

    2015-12-01

    Base Excision Repair (BER) is a conserved, intracellular DNA repair system that recognizes and removes chemically modified bases to insure genomic integrity and prevent mutagenesis. Aberrant BER has been tightly linked with a broad spectrum of human pathologies, such as several types of cancer, neurological degeneration, developmental abnormalities, immune dysfunction and aging. In the cell, BER must recognize and remove DNA lesions from the tightly condensed, protein-coated chromatin. Because chromatin is necessarily refractory to DNA metabolic processes, like transcription and replication, the compaction of the genomic material is also inhibitory to the repair systems necessary for its upkeep. Multiple ATP-dependent chromatin remodelling (ACR) complexes play essential roles in modulating the protein-DNA interactions within chromatin, regulating transcription and promoting activities of some DNA repair systems, including double-strand break repair and nucleotide excision repair. However, it remains unclear how BER operates in the context of chromatin, and if the chromatin remodelling processes that govern transcription and replication also actively regulate the efficiency of BER. In this review we highlight the emerging role of ACR in regulation of BER. PMID:26422134

  3. Snapshots: Chromatin Control of Viral Infection

    PubMed Central

    Knipe, David M.; Lieberman, Paul M.; Jung, Jae U.; McBride, Alison A.; Morris, Kevin V.; Ott, Melanie; Margolis, David; Nieto, Amelia; Nevels, Michael; Parks, Robin J.; Kristie, Thomas M.

    2012-01-01

    Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cell’s chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles. PMID:23217624

  4. Glycolytic metabolism influences global chromatin structure

    PubMed Central

    Liu, Xue-Song; Little, John B.; Yuan, Zhi-Min

    2015-01-01

    Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy. PMID:25784656

  5. Chromatin Computation

    PubMed Central

    Bryant, Barbara

    2012-01-01

    In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this “chromatin computer” to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal – and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines. PMID:22567109

  6. Lactobacillus reuteri modulates cytokines production in exhaled breath condensate of children with atopic dermatitis.

    PubMed

    Miniello, Vito Leonardo; Brunetti, Luigia; Tesse, Riccardina; Natile, Miria; Armenio, Lucio; Francavilla, Ruggiero

    2010-05-01

    We measured the concentration of interferon-gamma and interleukin-4 in the exhaled breath condensate of children with atopic and nonallergic dermatitis receiving a probiotic supplementation (Lactobacillus reuteri ATCC 55730) or placebo for 8 weeks. We demonstrated that the levels of these cytokines increased and decreased respectively only in atopic subjects receiving active treatment. Our data suggest that the oral administration of a specific probiotic strain in patients with atopic dermatitis can modulate in vivo the cytokine pattern at a different site from intestine. PMID:20639717

  7. Dark-dark solitons and modulational instability in miscible two-component Bose-Einstein condensates

    SciTech Connect

    Hoefer, M. A.; Chang, J. J.; Hamner, C.; Engels, P.

    2011-10-15

    We investigate the dynamics of two miscible superfluids experiencing fast counterflow in a narrow channel. The superfluids are formed by two distinguishable components of a trapped dilute-gas Bose-Einstein condensate (BEC). The onset of counterflow-induced modulational instability throughout the cloud is observed and shown to lead to the proliferation of dark-dark vector solitons. These solitons do not exist in single-component systems, exhibit intriguing beating dynamics, and can experience a transverse instability leading to vortex line structures. Experimental results and multidimensional numerical simulations are presented.

  8. HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

    PubMed Central

    Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

    2016-01-01

    The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

  9. Directed momentum current of Bose–Einstein condensate in the presence of spatially modulated nonlinear interaction

    NASA Astrophysics Data System (ADS)

    Zhao, Wen-Lei; Ding, Cai-Ying; Liu, Jie; Fu, Li-Bin

    2016-06-01

    We investigate the quantum transport dynamics of periodically delta-kicked Bose–Einstein condensate under the effect of spatially modulated nonlinear interactions. The spatial modulation frequency can dramatically affect the transport behaviors of the ultra-cold atoms. For odd frequency, the linear growth of the directed current is close to that of the noninteracting case for not very strong nonlinear interaction. Both the acceleration and the quantum state evolution gradually approach that of the noninteracting case with increasing frequency. For other values of frequency, a very weak nonlinear interaction can dramatically reduce the linear growth of the directed current. The quantum state evolution differs rapidly from that of the noninteracting case. The underlying dynamic mechanism is uncovered and some important implications are addressed.

  10. Quasi-two-dimensional Bose-Einstein condensates with spatially modulated cubic-quintic nonlinearities

    SciTech Connect

    Dai Chaoqing; Wang Dengshan; Wang Liangliang; Zhang Jiefang; Liu, W.M.

    2011-09-15

    We investigate exact nonlinear matter wave functions with odd and even parities in the framework of quasi-two-dimensional Bose-Einstein condensates (BECs) with spatially modulated cubic-quintic nonlinearities and harmonic potential. The existence condition for these exact solutions requires that the minimum energy eigenvalue of the corresponding linear Schroedinger equation with harmonic potential is the cutoff value of the chemical potential {lambda}. The competition between two-body and three-body interactions influences the energy of the localized state. For attractive two-body and three-body interactions, the larger the matter wave order number n, the larger the energy of the corresponding localized state. A linear stability analysis and direct simulations with initial white noise demonstrate that, for the same state (fixed n), increasing the number of atoms can add stability. A quasi-stable ground-state matter wave is also found for repulsive two-body and three-body interactions. We also discuss the experimental realization of these results in future experiments. These results are of particular significance to matter wave management in higher-dimensional BECs. - Highlights: > 2D Bose-Einstein condensates (BECs) with spatially modulated cubic-quintic nonlinearities and the harmonic potential are discussed. > 2D exact quantized nonlinear matter wave functions with the odd and even parities are obtained. > The 2D ground-state matter wave with attractive two-body and repulsive three-body interactions is stable. > Experimental realization of our results in future experiments is proposed.

  11. Dynamical stability of dipolar Bose-Einstein condensates with temporal modulation of the s-wave scattering length.

    PubMed

    Sabari, S; Jisha, Chandroth P; Porsezian, K; Brazhnyi, Valeriy A

    2015-09-01

    We study the stabilization properties of dipolar Bose-Einstein condensate by temporal modulation of short-range two-body interaction. Through both analytical and numerical methods, we analyze the mean-field Gross-Pitaevskii equation with short-range two-body and long-range, nonlocal, dipolar interaction terms. We derive the equation of motion and effective potential of the dipolar condensate by variational method. We show that there is an enhancement of the condensate stability due to the inclusion of dipolar interaction in addition to the two-body contact interaction. We also show that the stability of the dipolar condensate increases in the presence of time varying two-body contact interaction; the temporal modification of the contact interaction prevents the collapse of dipolar Bose-Einstein condensate. Finally we confirm the semi-analytical prediction through the direct numerical simulations of the governing equation. PMID:26465538

  12. Membrane-associated glucocorticoid activity is necessary for modulation of long-term memory via chromatin modification

    PubMed Central

    Roozendaal, Benno; Hernandez, Angelina; Cabrera, Sara M.; Hagewoud, Roelina; Malvaez, Melissa; Stefanko, Daniel P.; Haettig, Jakob; Wood, Marcelo A.

    2010-01-01

    Glucocorticoid hormones enhance the consolidation of long-term memory of emotionally arousing training experiences. This memory enhancement requires activation of the cAMP-dependent kinase pathway and the subsequent phosphorylation of cAMP response-element binding (CREB) protein. Here, we demonstrate that glucocorticoids enhance the consolidation of hippocampus-dependent and hippocampus-independent aspects of object recognition memory via chromatin modification. More specifically, systemic corticosterone increases histone acetylation, a form of chromatin modification, in both the hippocampus and insular cortex following training on an object recognition task. This led us to examine whether increasing histone acetylation via histone deacetylase (HDAC) inhibition enhances memory in a similar manner as corticosterone. We found a double dissociation between posttraining HDAC inhibitor infusion into the insular cortex and hippocampus on the enhancement of object recognition and object location memory, respectively. In determining the molecular pathway upstream of glucocorticoids’ effects on chromatin modification, we found that activation of membrane-associated glucocorticoid receptors (GRs) and the subsequent interaction between phospho-CREB and CREB-binding protein (CBP) appear to be necessary for glucocorticoids to enhance memory consolidation via chromatin modification. In contrast, mineralocorticoid receptors (MRs) do not appear to be involved. The findings also indicate that glucocorticoid activity has differential influences on hippocampus-dependent and hippocampus-independent components of memory for objects. PMID:20371824

  13. Correlated Spatio-Temporal Fluctuations in Chromatin Compaction States Characterize Stem Cells

    PubMed Central

    Talwar, Shefali; Kumar, Abhishek; Rao, Madan; Menon, Gautam I.; Shivashankar, G.V.

    2013-01-01

    Stem cells integrate signals from the microenvironment to generate lineage-specific gene expression programs upon differentiation. Undifferentiated cell nuclei are easily deformable, with an active transcriptome, whereas differentiated cells have stiffer nuclei and condensed chromatin. Chromatin organization in the stem cell state is known to be highly dynamic but quantitative characterizations of its plasticity are lacking. Using fluorescence imaging, we study the spatio-temporal dynamics of nuclear architecture and chromatin compaction in mouse embryonic stem (ES) cells and differentiated states. Individual ES cells exhibit a relatively narrow variation in chromatin compaction, whereas primary mouse embryonic fibroblasts (PMEF) show broad distributions. However, spatial correlations in chromatin compaction exhibit an emergent length scale in PMEFs, although they are unstructured and longer ranged in ES cells. We provide evidence for correlated fluctuations with large amplitude and long intrinsic timescales, including an oscillatory component, in both chromatin compaction and nuclear area in ES cells. Such fluctuations are largely frozen in PMEF. The role of actin and Lamin A/C in modulating these fluctuations is described. A simple theoretical formulation reproduces the observed dynamics. Our results suggest that, in addition to nuclear plasticity, correlated spatio-temporal structural fluctuations of chromatin in undifferentiated cells characterize the stem cell state. PMID:23442906

  14. Poly(ADP-Ribosyl)ation Is Required to Modulate Chromatin Changes at c-MYC Promoter during Emergence from Quiescence

    PubMed Central

    Battistelli, Cecilia; Ciotti, Agnese; Amati, Paolo; Maione, Rossella

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation. PMID:25047032

  15. Theta and Alpha Band Modulations Reflect Error-Related Adjustments in the Auditory Condensation Task

    PubMed Central

    Novikov, Nikita A.; Bryzgalov, Dmitri V.; Chernyshev, Boris V.

    2015-01-01

    Error commission leads to adaptive adjustments in a number of brain networks that subserve goal-directed behavior, resulting in either enhanced stimulus processing or increased motor threshold depending on the nature of errors committed. Here, we studied these adjustments by analyzing post-error modulations of alpha and theta band activity in the auditory version of the two-choice condensation task, which is highly demanding for sustained attention while involves no inhibition of prepotent responses. Errors were followed by increased frontal midline theta (FMT) activity, as well as by enhanced alpha band suppression in the parietal and the left central regions; parietal alpha suppression correlated with the task performance, left central alpha suppression correlated with the post-error slowing, and FMT increase correlated with both behavioral measures. On post-error correct trials, left-central alpha band suppression started earlier before the response, and the response was followed by weaker FMT activity, as well as by enhanced alpha band suppression distributed over the entire scalp. These findings indicate that several separate neuronal networks are involved in post-error adjustments, including the midfrontal performance monitoring network, the parietal attentional network, and the sensorimotor network. Supposedly, activity within these networks is rapidly modulated after errors, resulting in optimization of their functional state on the subsequent trials, with corresponding changes in behavioral measures. PMID:26733266

  16. Deoxyribonuclease II as a probe to sequence-specific chromatin organization: preferential cleavage in the 72 bp modulator sequence of SV40 minichromosome.

    PubMed Central

    Shakhov, A N; Nedospasov, S A; Georgiev, G P

    1982-01-01

    T prove sequence-specific chromatin structure of SV40 minichromosome, we further modified previously described hybridization mapping. Actually (i) the digestion patterns by two nucleases (micrococcal and DNAase II) were compared and (ii) the kinetics of nuclease digestion was analyzed from early time points when only a fraction of minichromosomes was cleaved once to longer digestions when oligo- and mononucleosomal bands appeared. DNAase II is shown to possess certain sequence specificity different from that of micrococcal nuclease. The major finding is that DNAase II preferentially cleaves the SV40 minichromosome at a distinct region of the genome known as 72 bp modulator element. Other hypersensitive sites are located near the replication origin and T-ag binding site II and also near BamHI site where termination of replication and "late" transcription occurs. Micrococcal nuclease splits the BglI-Hpaii region in a different manner. Images PMID:6287427

  17. Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators.

    PubMed

    Yan, Qinghong; Weyn-Vanhentenryck, Sebastien M; Wu, Jie; Sloan, Steven A; Zhang, Ye; Chen, Kenian; Wu, Jia Qian; Barres, Ben A; Zhang, Chaolin

    2015-03-17

    Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome. PMID:25737549

  18. Quasi-two-dimensional Bose-Einstein condensates with spatially modulated cubic-quintic nonlinearities

    NASA Astrophysics Data System (ADS)

    Dai, Chao-Qing; Wang, Deng-Shan; Wang, Liang-Liang; Zhang, Jie-Fang; Liu, W. M.

    2011-09-01

    We investigate exact nonlinear matter wave functions with odd and even parities in the framework of quasi-two-dimensional Bose-Einstein condensates (BECs) with spatially modulated cubic-quintic nonlinearities and harmonic potential. The existence condition for these exact solutions requires that the minimum energy eigenvalue of the corresponding linear Schrödinger equation with harmonic potential is the cutoff value of the chemical potential λ. The competition between two-body and three-body interactions influences the energy of the localized state. For attractive two-body and three-body interactions, the larger the matter wave order number n, the larger the energy of the corresponding localized state. A linear stability analysis and direct simulations with initial white noise demonstrate that, for the same state (fixed n), increasing the number of atoms can add stability. A quasi-stable ground-state matter wave is also found for repulsive two-body and three-body interactions. We also discuss the experimental realization of these results in future experiments. These results are of particular significance to matter wave management in higher-dimensional BECs.

  19. 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

  20. EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

    PubMed Central

    Golovnin, Anton; Melnikova, Larisa; Shapovalov, Igor; Kostyuchenko, Margarita; Georgiev, Pavel

    2015-01-01

    Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization. PMID:26489095

  1. Quiescent Saccharomyces cerevisiae forms telomere hyperclusters at the nuclear membrane vicinity through a multifaceted mechanism involving Esc1, the Sir complex, and chromatin condensation.

    PubMed

    Laporte, Damien; Courtout, Fabien; Tollis, Sylvain; Sagot, Isabelle

    2016-06-15

    Like other eukaryotes, Saccharomyces cerevisiae spatially organizes its chromosomes within the nucleus. In G1 phase, the yeast's 32 telomeres are clustered into 6-10 foci that dynamically interact with the nuclear membrane. Here we show that, when cells leave the division cycle and enter quiescence, telomeres gather into two to three hyperclusters at the nuclear membrane vicinity. This localization depends on Esc1 but not on the Ku proteins. Telomere hypercluster formation requires the Sir complex but is independent of the nuclear microtubule bundle that specifically assembles in quiescent cells. Importantly, mutants deleted for the linker histone H1 Hho1 or defective in condensin activity or affected for histone H4 Lys-16 deacetylation are impaired, at least in part, for telomere hypercluster formation in quiescence, suggesting that this process involves chromosome condensation. Finally, we establish that telomere hypercluster formation is not necessary for quiescence establishment, maintenance, and exit, raising the question of the physiological raison d'être of this nuclear reorganization. PMID:27122604

  2. Quiescent Saccharomyces cerevisiae forms telomere hyperclusters at the nuclear membrane vicinity through a multifaceted mechanism involving Esc1, the Sir complex, and chromatin condensation

    PubMed Central

    Laporte, Damien; Courtout, Fabien; Tollis, Sylvain; Sagot, Isabelle

    2016-01-01

    Like other eukaryotes, Saccharomyces cerevisiae spatially organizes its chromosomes within the nucleus. In G1 phase, the yeast’s 32 telomeres are clustered into 6–10 foci that dynamically interact with the nuclear membrane. Here we show that, when cells leave the division cycle and enter quiescence, telomeres gather into two to three hyperclusters at the nuclear membrane vicinity. This localization depends on Esc1 but not on the Ku proteins. Telomere hypercluster formation requires the Sir complex but is independent of the nuclear microtubule bundle that specifically assembles in quiescent cells. Importantly, mutants deleted for the linker histone H1 Hho1 or defective in condensin activity or affected for histone H4 Lys-16 deacetylation are impaired, at least in part, for telomere hypercluster formation in quiescence, suggesting that this process involves chromosome condensation. Finally, we establish that telomere hypercluster formation is not necessary for quiescence establishment, maintenance, and exit, raising the question of the physiological raison d’être of this nuclear reorganization. PMID:27122604

  3. Bhas 42 cell transformation activity of cigarette smoke condensate is modulated by selenium and arsenic.

    PubMed

    Han, Sung Gu; Pant, Kamala; Bruce, Shannon W; Gairola, C Gary

    2016-04-01

    Cigarette smoking remains a major health risk worldwide. Development of newer tobacco products requires the use of quantitative toxicological assays. Recently, v-Ha-ras transfected BALB/c3T3 (Bhas 42) cell transformation assay was established that simulates the two-stage animal tumorigenesis model and measures tumor initiating and promoting activities of chemicals. The present study was performed to assess the feasibility of using this Bhas 42 cell transformation assay to determine the initiation and promotion activities of cigarette smoke condensate (CSC) and its water soluble fraction. Further, the modulating effects of selenium and arsenic on cigarette smoke-induced cell transformation were investigated. Dimethyl sulfoxide (DMSO) and water extracts of CSC (CSC-D and CSC-W, respectively) were tested at concentrations of 2.5-40 µg mL(-1) in the initiation or promotion assay formats. Initiation protocol of the Bhas 42 assay showed a 3.5-fold increase in transformed foci at 40 µg mL(-1) of CSC-D but not CSC-W. The promotion phase of the assay yielded a robust dose response with CSC-D (2.5-40 µg mL(-1)) and CSC-W (20-40 µg mL(-1)). Preincubation of cells with selenium (100 nM) significantly reduced CSC-induced increase in cell transformation in initiation assay. Co-treatment of cells with a sub-toxic dose of arsenic significantly enhanced cell transformation activity of CSC-D in promotion assay. The results suggest a presence of both water soluble and insoluble tumor promoters in CSC, a role of oxidative stress in CSC-induced cell transformation, and usefulness of Bhas 42 cell transformation assay in comparing tobacco product toxicities and in studying the mechanisms of tobacco carcinogenesis. PMID:26924598

  4. Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

    NASA Technical Reports Server (NTRS)

    Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

    1996-01-01

    A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

  5. Chromatin Compaction Protects Genomic DNA from Radiation Damage

    PubMed Central

    Takata, Hideaki; Hanafusa, Tomo; Mori, Toshiaki; Shimura, Mari; Iida, Yutaka; Ishikawa, Kenichi; Yoshikawa, Kenichi; Yoshikawa, Yuko; Maeshima, Kazuhiro

    2013-01-01

    Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity. PMID:24130727

  6. Chromosome condensation and decondensation during mitosis.

    PubMed

    Antonin, Wolfram; Neumann, Heinz

    2016-06-01

    During eukaryotic cell division, nuclear chromatin undergoes marked changes with respect to shape and degree of compaction. Although already significantly compacted during interphase, upon entry into mitosis chromatin further condenses and individualizes to discrete chromosomes that are captured and moved independently by the mitotic spindle apparatus. Once segregated by the spindle, chromatin decondenses to re-establish its interphase structure competent for DNA replication and transcription. Although cytologically described a long time ago, the underlying molecular mechanisms of mitotic chromatin condensation and decondensation are still ill-defined. Here we summarize our current knowledge of mitotic chromatin restructuring and recent progress in the field. PMID:26895139

  7. Histone H3 phosphorylation – A versatile chromatin modification for different occasions

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2012-01-01

    Post-translation modifications of histones modulate the accessibility and transcriptional competence of specific chromatin regions within the eukaryotic genome. Phosphorylation of histone H3 is unique in the sense that it associates on one hand with open chromatin during gene activation and marks on the other hand highly condensed chromatin during mitosis. Phosphorylation of serine residues at histone H3 is a highly dynamic process that creates together with acetylation and methylation marks at neighboring lysine residues specific combinatorial patterns that are read by specific detector proteins. In this review we describe the importance of different histone H3 phosphorylation marks for chromatin condensation during mitosis. In addition, we review the signals that trigger histone H3 phosphorylation and the factors that control this reversible modification during interphase and mediate the biological readout of the signal. Finally, we discuss different models describing the role of histone H3 phosphorylation in the activation of transcription of poised genes or by transient derepression of epigenetically silenced genes. We propose that histone H3 phosphorylation in the context with lysine methylation might temporarily relieve the silencing of specific genes without affecting the epigenetic memory. PMID:22564826

  8. The Chromatin Regulator DMAP1 Modulates Activity of the Nuclear Factor κB (NF-κB) Transcription Factor Relish in the Drosophila Innate Immune Response*

    PubMed Central

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A.

    2014-01-01

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-κB family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-κB protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  9. The chromatin regulator DMAP1 modulates activity of the nuclear factor B (NF-B) transcription factor Relish in the Drosophila innate immune response.

    PubMed

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A

    2014-07-25

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-B family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-B protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  10. The Double-Bromodomain Proteins Bdf1 and Bdf2 Modulate Chromatin Structure to Regulate S-Phase Stress Response in Schizosaccharomyces pombe

    PubMed Central

    Garabedian, Mikael V.; Noguchi, Chiaki; Ziegler, Melissa A.; Das, Mukund M.; Singh, Tanu; Harper, Logan J.; Leman, Adam R.; Khair, Lyne; Moser, Bettina A.; Nakamura, Toru M.; Noguchi, Eishi

    2012-01-01

    Bromodomain proteins bind acetylated histones to regulate transcription. Emerging evidence suggests that histone acetylation plays an important role in DNA replication and repair, although its precise mechanisms are not well understood. Here we report studies of two double bromodomain-containing proteins, Bdf1 and Bdf2, in fission yeast. Loss of Bdf1 or Bdf2 led to a reduction in the level of histone H4 acetylation. Both bdf1Δ and bdf2Δ cells showed sensitivity to DNA damaging agents, including camptothecin, that cause replication fork breakage. Consistently, Bdf1 and Bdf2 were important for recovery of broken replication forks and suppression of DNA damage. Surprisingly, deletion of bdf1 or bdf2 partially suppressed sensitivity of various checkpoint mutants including swi1Δ, mrc1Δ, cds1Δ, crb2Δ, chk1Δ, and rad3Δ, to hydroxyurea, a compound that stalls replication forks and activates the Cds1-dependent S-phase checkpoint. This suppression was not due to reactivation of Cds1. Instead, we found that bdf2 deletion alleviates DNA damage accumulation caused by defects in the DNA replication checkpoint. We also show that hydroxyurea sensitivity of mrc1Δ and swi1Δ was suppressed by mutations in histone H4 acetyltransferase subunits or histone H4. These results suggest that the double bromodomain-containing proteins modulate chromatin structure to coordinate DNA replication and S-phase stress response. PMID:22095079

  11. Valproic acid modulates brain plasticity through epigenetic chromatin remodeling in the blind rat: implications for human sight recovery.

    PubMed

    Fetter-Pruneda, I; Martínez-Méndez, R; Olivos-Cisneros, L; Diaz, D; Padilla-Cortés, P; Báez-Saldaña, A; Gutiérrez-Ospina, G

    2011-01-01

    Blindness is a pervasive sensory condition that imposes diverse difficulties to carry on with activities of daily living. In blind individuals, the brain is subjected to a large scale reorganization characterized by expanded cortical territories associated with somatosensory and auditory functions and the recruitment of the former visual areas to perform bimodal somatosensory and auditory integration. This poses obstacles to efforts aimed at reassigning visual functions to the recruited visual cortex in the blind, especially after the end of the ontogentic sensitive period. Devising pharmacological measures to modulate the magnitude of brain plasticity could improve our chances of recovering visual functions in the blind. Here, by using the primary somatosensory cortex (S1) in the rat as a working model, we showed that valproic acid administered through the mother's milk prevents cortical reorganization in blinded rats by delaying neuronal histone de-acetylation. These results suggest that in the future, we might be able to devise epigenetic pharmacological measures that could improve our chances of reassigning visual functions to the once deprived former visual cortex in the blind, by modulating the magnitude of brain plasticity during critical times of development. PMID:22423589

  12. A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

    PubMed Central

    Olguin-Lamas, Alejandro; Madec, Edwige; Hovasse, Agnes; Werkmeister, Elisabeth; Callebaut, Isabelle; Slomianny, Christian; Delhaye, Stephane; Mouveaux, Thomas; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain; Tomavo, Stanislas

    2011-01-01

    In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome

  13. Modulating the amplitude of dark soliton by scattering-length management in Bose-Einstein condensates

    NASA Astrophysics Data System (ADS)

    Zhang, W. X.; Wang, D. L.; He, Z. M.; Wang, F. J.; Ding, J. W.

    2008-06-01

    We present a family of soliton solutions of the quasi-one-dimensional Bose-Einstein condensates with time-dependent scattering length, by developing multiple-scale method combined with truncated Painlevé expansion. Then, by numerical calculating the solutions, it is shown that there exhibit two types of dark solitons-black soliton (the zero minimum amplitude at its center) and gray soliton (the minimum density does not drop to zero) in a repulsive condensate. Furthermore, we propose experimental protocols to realize the exchange between black and gray solitons by varying the scattering length via the Feshbach resonance in currently experimental conditions.

  14. 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.

  15. A Macrohistone Variant Links Dynamic Chromatin Compaction to BRCA1-Dependent Genome Maintenance

    PubMed Central

    Khurana, Simran; Kruhlak, Michael J.; Kim, Jeongkyu; Tran, Andy D.; Liu, Jinping; Nyswaner, Katherine; Shi, Lei; Jailwala, Parthav; Sung, Myong-Hee; Hakim, Ofir; Oberdoerffer, Philipp

    2014-01-01

    SUMMARY Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate repair outcome and genomic integrity. The factors that regulate this process remain poorly understood. Here, we identify two repressive chromatin components, the macrohistone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together direct the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ataxia telangiectasia mutated (ATM)-dependent accumulation of both proteins at DSBs in order to promote DSB-flanking H3K9 dimethylation. Remarkably, loss of macroH2A1 or PRDM2, as well as experimentally induced chromatin decondensation, impairs the retention of BRCA1, but not 53BP1, at DSBs. As a result, mac-roH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair, and the resistance to poly(ADP-ribose) polymerase (PARP) inhibition—all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance. PMID:25131201

  16. 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

  17. Stationary States and Modulational Instability of Coupled Two-Component Bose-Einstein Condensates in a Ring Trap

    NASA Astrophysics Data System (ADS)

    Deng, Hai-Ming; Zhong, Hong-Hua; Huang, Jia-Hao; Dai, Hui; Yao, Min; Huang, Xiao-Yi

    2015-08-01

    We investigate modulational instability (MI) of a coupled two-component Bose-Einstein condensates in a rotating ring trap. The excitation spectrum and the MI condition of the system are presented analytically. We find that the coupling between the two components strongly modifies the MI condition, and the MI condition is phase-dependent. Furthermore, we discuss the effect of MI on both density excitation and spin excitation. If the inter- and intra-component interaction strengths are all equal, the MI causes density excitation but not spin excitation, and if the inter- and intra-component interaction strengths are different, the MI causes both density excitation and spin excitation. Our results provide a promising approach for controlling the stability and excitation of a rotating two-component Bose-Einstein condensates by modulating its coupling strength and interaction strength. Supported by the National Natural Science Foundation of China under Grant No. 11465008, the Hunan Provincial Natural Science Foundation under Grant No. 2015JJ2114, the Scientific Research Fund of Hunan Provincial Education Department under Grant Nos. 14A118, 13C881, Science and Technology Innovative Research Team in Higher Educational Instituions of Hunan Province, and Science Research Foundation of Xiangnan University under Grant No. 2012-126(41)

  18. 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

  19. 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. PMID:25386906

  20. Computational strategies to address chromatin structure problems.

    PubMed

    Perišić, Ognjen; Schlick, Tamar

    2016-01-01

    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. PMID:27345617

  1. Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility

    PubMed Central

    Castillo, Judit; Estanyol, Josep Maria; Ballescà, Josep Lluis; Oliva, Rafael

    2015-01-01

    The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo. PMID:25926607

  2. Phosphorylation of histone variant regions in chromatin: unlocking the linker?

    PubMed

    Green, G R

    2001-01-01

    Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented. PMID:11467741

  3. Integrable model for density-modulated quantum condensates: Solitons passing through a soliton lattice

    NASA Astrophysics Data System (ADS)

    Takahashi, Daisuke A.

    2016-06-01

    An integrable model possessing inhomogeneous ground states is proposed as an effective model of nonuniform quantum condensates such as supersolids and Fulde-Ferrell-Larkin-Ovchinnikov superfluids. The model is a higher-order analog of the nonlinear Schrödinger equation. We derive an n -soliton solution via the inverse scattering theory with elliptic-functional background and reveal various kinds of soliton dynamics such as dark soliton billiards, dislocations, gray solitons, and envelope solitons. We also provide the exact bosonic and fermionic quasiparticle eigenstates and show their tunneling phenomena. The solutions are expressed by a determinant of theta functions.

  4. Integrable model for density-modulated quantum condensates: Solitons passing through a soliton lattice.

    PubMed

    Takahashi, Daisuke A

    2016-06-01

    An integrable model possessing inhomogeneous ground states is proposed as an effective model of nonuniform quantum condensates such as supersolids and Fulde-Ferrell-Larkin-Ovchinnikov superfluids. The model is a higher-order analog of the nonlinear Schrödinger equation. We derive an n-soliton solution via the inverse scattering theory with elliptic-functional background and reveal various kinds of soliton dynamics such as dark soliton billiards, dislocations, gray solitons, and envelope solitons. We also provide the exact bosonic and fermionic quasiparticle eigenstates and show their tunneling phenomena. The solutions are expressed by a determinant of theta functions. PMID:27415270

  5. Histone acetylation: a switch between repressive and permissive chromatin

    PubMed Central

    Eberharter, Anton; Becker, Peter B.

    2002-01-01

    The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. PMID:11882541

  6. Localized spatially nonlinear matter waves in atomic-molecular Bose-Einstein condensates with space-modulated nonlinearity.

    PubMed

    Yao, Yu-Qin; Li, Ji; Han, Wei; Wang, Deng-Shan; Liu, Wu-Ming

    2016-01-01

    The intrinsic nonlinearity is the most remarkable characteristic of the Bose-Einstein condensates (BECs) systems. Many studies have been done on atomic BECs with time- and space- modulated nonlinearities, while there is few work considering the atomic-molecular BECs with space-modulated nonlinearities. Here, we obtain two kinds of Jacobi elliptic solutions and a family of rational solutions of the atomic-molecular BECs with trapping potential and space-modulated nonlinearity and consider the effect of three-body interaction on the localized matter wave solutions. The topological properties of the localized nonlinear matter wave for no coupling are analysed: the parity of nonlinear matter wave functions depends only on the principal quantum number n, and the numbers of the density packets for each quantum state depend on both the principal quantum number n and the secondary quantum number l. When the coupling is not zero, the localized nonlinear matter waves given by the rational function, their topological properties are independent of the principal quantum number n, only depend on the secondary quantum number l. The Raman detuning and the chemical potential can change the number and the shape of the density packets. The stability of the Jacobi elliptic solutions depends on the principal quantum number n, while the stability of the rational solutions depends on the chemical potential and Raman detuning. PMID:27403634

  7. Localized spatially nonlinear matter waves in atomic-molecular Bose-Einstein condensates with space-modulated nonlinearity

    PubMed Central

    Yao, Yu-Qin; Li, Ji; Han, Wei; Wang, Deng-Shan; Liu, Wu-Ming

    2016-01-01

    The intrinsic nonlinearity is the most remarkable characteristic of the Bose-Einstein condensates (BECs) systems. Many studies have been done on atomic BECs with time- and space- modulated nonlinearities, while there is few work considering the atomic-molecular BECs with space-modulated nonlinearities. Here, we obtain two kinds of Jacobi elliptic solutions and a family of rational solutions of the atomic-molecular BECs with trapping potential and space-modulated nonlinearity and consider the effect of three-body interaction on the localized matter wave solutions. The topological properties of the localized nonlinear matter wave for no coupling are analysed: the parity of nonlinear matter wave functions depends only on the principal quantum number n, and the numbers of the density packets for each quantum state depend on both the principal quantum number n and the secondary quantum number l. When the coupling is not zero, the localized nonlinear matter waves given by the rational function, their topological properties are independent of the principal quantum number n, only depend on the secondary quantum number l. The Raman detuning and the chemical potential can change the number and the shape of the density packets. The stability of the Jacobi elliptic solutions depends on the principal quantum number n, while the stability of the rational solutions depends on the chemical potential and Raman detuning. PMID:27403634

  8. Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.

    PubMed

    Noveen, A; Jiang, T X; Chuong, C M

    1995-10-01

    The molecular signaling of secondary induction is a fundamental process in organogenesis during embryonic development. To study the signal transduction pathways involved, we used developing chicken skin as a model and focused on the roles of intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of forming dermis around H and H stage 30. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. The presence and activity of PKC, PKA, and P-CREB in developing chicken skin are further characterized by immunoblot, kinase activity, and gel shift assays. To explore their physiological significance, embryonic chicken dorsal skin explants were treated with different modulators in medium or in beads for localized effects. The results showed that PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. We observed that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented. PMID:7556946

  9. 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.

  10. Condensing polarization mode dispersion using CSRZ-DQPSK modulation in WDM-RoF-PON system

    NASA Astrophysics Data System (ADS)

    Sanofer, P. Fathima; Julus, L. Jerart; Vijayarangan, V.

    2015-06-01

    Differential Quadrature Phase Shift Keying (DQPSK) is considered as an acceptable modulation technique for long-haul high speed communication owing to its ability to diminish the Polarization Mode Dispersion (PMD), non-linear effects and has constant envelope over the communication. The assessment includes Carrier Suppressed Return to Zero (CSRZ) modulation format. The combination of CSRZ and DQPSK techniques would result in a high bit rate with better Bit Error Rate (BER) in Wavelength Division Multiplexing - Passive Optical Network (WDM-PON) setup. The Radio Frequency (RF) signal is obtained finally for wireless service which can be assessed. . The optical communication system is expanded by comprising Polarization Multiplexing (PM) which improves the user scope and the spectral efficiency. The goal is to experimentally analyze the performances of CSRZ-DQPSK modulated non-coherent receiver for wire and RoF (Radio over Fiber) system for wireless service. Also, equal amount of power is provided for both the services. The PMD is reduced both the services. The Proposed system can provide flexible, cost-effective and high bandwidth utilization with reduced BER.

  11. Organization of higher-level chromatin structures (chromomere, chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization.

    PubMed

    Sheval, E V; Prusov, A N; Kireev, I I; Fais, D; Polyakov, V Yu

    2002-01-01

    The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei. PMID:12127937

  12. The landscape of accessible chromatin in mammalian preimplantation embryos.

    PubMed

    Wu, Jingyi; Huang, Bo; Chen, He; Yin, Qiangzong; Liu, Yang; Xiang, Yunlong; Zhang, Bingjie; Liu, Bofeng; Wang, Qiujun; Xia, Weikun; Li, Wenzhi; Li, Yuanyuan; Ma, Jing; Peng, Xu; Zheng, Hui; Ming, Jia; Zhang, Wenhao; Zhang, Jing; Tian, Geng; Xu, Feng; Chang, Zai; Na, Jie; Yang, Xuerui; Xie, Wei

    2016-06-30

    In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development. PMID:27309802

  13. Chromatin immunoprecipitation and an open chromatin assay in zebrafish erythrocytes.

    PubMed

    Yang, S; Ott, C J; Rossmann, M P; Superdock, M; Zon, L I; Zhou, Y

    2016-01-01

    Zebrafish is an excellent genetic and developmental model for the study of vertebrate development and disease. Its ability to produce an abundance of transparent, externally developed embryos has facilitated large-scale genetic and chemical screens for the identification of critical genes and chemical factors that modulate developmental pathways. These studies can have profound implications for the diagnosis and treatment of a variety of human diseases. Recent advancements in molecular and genomic studies have provided valuable tools and resources for comprehensive and high-resolution analysis of epigenomes during cell specification and lineage differentiation throughout development. In this chapter, we describe two simple methods to evaluate protein-DNA interaction and chromatin architecture in erythrocytes from adult zebrafish. These are chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) and an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). These techniques, together with gene expression profiling, are useful for analyzing epigenomic regulation of cell specification, differentiation, and function during zebrafish development in both normal and disease models. PMID:27443937

  14. 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.

  15. Gearing up chromatin

    PubMed Central

    Mandemaker, Imke K; Vermeulen, Wim; Marteijn, Jurgen A

    2014-01-01

    During transcription, RNA polymerase may encounter DNA lesions, which causes stalling of transcription. To overcome the RNA polymerase blocking lesions, the transcribed strand is repaired by a dedicated repair mechanism, called transcription coupled nucleotide excision repair (TC-NER). After repair is completed, it is essential that transcription restarts. So far, the regulation and exact molecular mechanism of this transcriptional restart upon genotoxic damage has remained elusive. Recently, three different chromatin remodeling factors, HIRA, FACT, and Dot1L, were identified to stimulate transcription restart after DNA damage. These factors either incorporate new histones or establish specific chromatin marks that will gear up the chromatin to subsequently promote transcription recovery. This adds a new layer to the current model of chromatin remodeling necessary for repair and indicates that this specific form of transcription, i.e., the transcriptional restart upon DNA damage, needs specific chromatin remodeling events. PMID:24809693

  16. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter.

    PubMed

    Visser, Mijke; Kayser, Manfred; Palstra, Robert-Jan

    2012-03-01

    Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases. PMID:22234890

  17. Minor Groove Binder Distamycin Remodels Chromatin but Inhibits Transcription

    PubMed Central

    Majumder, Parijat; Banerjee, Amrita; Shandilya, Jayasha; Senapati, Parijat; Chatterjee, Snehajyoti; Kundu, Tapas K.; Dasgupta, Dipak

    2013-01-01

    The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as “chromatin remodeling”. In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance. PMID:23460895

  18. Functional interplay between histone H1 and HMG proteins in chromatin.

    PubMed

    Postnikov, Yuri V; Bustin, Michael

    2016-03-01

    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

  19. 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

  20. Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

    PubMed Central

    Linnemann, Amelia K.; Krawetz, Stephen A.

    2010-01-01

    Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

  1. Prenucleosomes and Active Chromatin

    PubMed Central

    Khuong, Mai T.; Fei, Jia; Ishii, Haruhiko; Kadonaga, James T.

    2016-01-01

    Chromatin consists of nucleosomes as well as nonnucleosomal histone-containing particles. Here we describe the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with ~80 bp DNA. Prenucleosomes are formed rapidly upon the deposition of histones onto DNA and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Different lines of evidence reveal that there are prenucleosome-sized DNA-containing particles with histones in the upstream region of active promoters. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes, and H3K56 acetylation is found at active promoters and enhancers. These findings therefore suggest that there may be prenucleosomes or prenucleosome-like particles in the upstream region of active promoters. More generally, we postulate that prenucleosomes or prenucleosome-like particles are present at dynamic chromatin, whereas canonical nucleosomes are at static chromatin. PMID:26767995

  2. Chromatin deregulation in disease.

    PubMed

    Mirabella, Anne C; Foster, Benjamin M; Bartke, Till

    2016-03-01

    The regulation of chromatin by epigenetic mechanisms plays a central role in gene expression and is essential for development and maintenance of cell identity and function. Aberrant chromatin regulation is observed in many diseases where it leads to defects in epigenetic gene regulation resulting in pathological gene expression programmes. These defects are caused by inherited or acquired mutations in genes encoding enzymes that deposit or remove DNA and histone modifications and that shape chromatin architecture. Chromatin deregulation often results in neurodevelopmental disorders and intellectual disabilities, frequently linked to physical and developmental abnormalities, but can also cause neurodegenerative diseases, immunodeficiency, or muscle wasting syndromes. Epigenetic diseases can either be of monogenic origin or manifest themselves as complex multifactorial diseases such as in congenital heart disease, autism spectrum disorders, or cancer in which mutations in chromatin regulators are contributing factors. The environment directly influences the epigenome and can induce changes that cause or predispose to diseases through risk factors such as stress, malnutrition or exposure to harmful chemicals. The plasticity of chromatin regulation makes targeting the enzymatic machinery an attractive strategy for therapeutic intervention and an increasing number of small molecule inhibitors against a variety of epigenetic regulators are in clinical use or under development. In this review, we will give an overview of the molecular lesions that underlie epigenetic diseases, and we will discuss the impact of the environment and prospects for epigenetic therapies. PMID:26188466

  3. Nucleoporins and chromatin metabolism.

    PubMed

    Ptak, Christopher; Wozniak, Richard W

    2016-06-01

    Mounting evidence has implicated a group of proteins termed nucleoporins, or Nups, in various processes that regulate chromatin structure and function. Nups were first recognized as building blocks for nuclear pore complexes, but several members of this group of proteins also reside in the cytoplasm and within the nucleus. Moreover, many are dynamic and move between these various locations. Both at the nuclear envelope, as part of nuclear pore complexes, and within the nucleoplasm, Nups interact with protein complexes that function in gene transcription, chromatin remodeling, DNA repair, and DNA replication. Here, we review recent studies that provide further insight into the molecular details of these interactions and their role in regulating the activity of chromatin modifying factors. PMID:27085162

  4. Chromatin signatures of cancer

    PubMed Central

    Morgan, Marc A.; Shilatifard, Ali

    2015-01-01

    Changes in the pattern of gene expression play an important role in allowing cancer cells to acquire their hallmark characteristics, while genomic instability enables cells to acquire genetic alterations that promote oncogenesis. Chromatin plays central roles in both transcriptional regulation and the maintenance of genomic stability. Studies by cancer genome consortiums have identified frequent mutations in genes encoding chromatin regulatory factors and histone proteins in human cancer, implicating them as major mediators in the pathogenesis of both hematological malignancies and solid tumors. Here, we review recent advances in our understanding of the role of chromatin in cancer, focusing on transcriptional regulatory complexes, enhancer-associated factors, histone point mutations, and alterations in heterochromatin-interacting factors. PMID:25644600

  5. Studies on chromatin. II. Isolation and characterization of chromatin subunits.

    PubMed Central

    Bakayev, V V; Melnickov, A A; Osicka, V D; Varshausky, A J

    1975-01-01

    Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described. Images PMID:1178523

  6. 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,…

  7. 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. PMID:23751185

  8. 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

  9. 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. PMID:26042538

  10. CPF-Associated Phosphatase Activity Opposes Condensin-Mediated Chromosome Condensation

    PubMed Central

    Vanoosthuyse, Vincent; Legros, Pénélope; van der Sar, Sjaak J. A.; Yvert, Gaël; Toda, Kenji; Le Bihan, Thierry; Watanabe, Yoshinori; Hardwick, Kevin; Bernard, Pascal

    2014-01-01

    Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3′ end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1Dis2 with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72. PMID:24945319

  11. Aging-related chromatin defects via loss of the NURD complex

    PubMed Central

    Pegoraro, Gianluca; Kubben, Nard; Wickert, Ute; Göhler, Heike; Hoffmann, Katrin; Misteli, Tom

    2009-01-01

    Physiological and premature aging are characterized by multiple defects in chromatin structure and accumulation of persistent DNA damage. Here we identify the NURD remodeling complex as a key modulator of these aging-associated chromatin defects. We demonstrate loss of several NURD components during premature and normal aging and we find aging-associated reduction of HDAC1 activity. Silencing of individual NURD subunits recapitulates some chromatin defects associated with aging and we provide evidence that structural chromatin defects precede DNA damage accumulation. These results outline a molecular mechanism for chromatin defects during aging. PMID:19734887

  12. Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells

    PubMed Central

    Heo, Su-Jin; Thorpe, Stephen D.; Driscoll, Tristan P.; Duncan, Randall L.; Lee, David A.; Mauck, Robert L.

    2015-01-01

    Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification. PMID:26592929

  13. 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

  14. Chromatin remodeling effects on enhancer activity.

    PubMed

    García-González, Estela; Escamilla-Del-Arenal, Martín; Arzate-Mejía, Rodrigo; Recillas-Targa, Félix

    2016-08-01

    During organism development, a diversity of cell types emerges with disparate, yet stable profiles of gene expression with distinctive cellular functions. In addition to gene promoters, the genome contains enhancer regulatory sequences, which are implicated in cellular specialization by facilitating cell-type and tissue-specific gene expression. Enhancers are DNA binding elements characterized by highly sophisticated and various mechanisms of action allowing for the specific interaction of general and tissue-specific transcription factors (TFs). However, eukaryotic organisms package their genetic material into chromatin, generating a physical barrier for TFs to interact with their cognate sequences. The ability of TFs to bind DNA regulatory elements is also modulated by changes in the chromatin structure, including histone modifications, histone variants, ATP-dependent chromatin remodeling, and the methylation status of DNA. Furthermore, it has recently been revealed that enhancer sequences are also transcribed into a set of enhancer RNAs with regulatory potential. These interdependent processes act in the context of a complex network of chromatin interactions, which together contributes to a renewed vision of how gene activation is coordinated in a cell-type-dependent manner. In this review, we describe the interplay between genetic and epigenetic aspects associated with enhancers and discuss their possible roles on enhancer function. PMID:27026300

  15. 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.

  16. 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.

  17. Ectopically tethered CP190 induces large-scale chromatin decondensation

    PubMed Central

    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. PMID:24472778

  18. Interplay of RNA Pol IV and ROS1 during post-embryonic 5S rDNA chromatin remodeling.

    PubMed

    Douet, Julien; Blanchard, Bertrand; Cuvillier, Claudine; Tourmente, Sylvette

    2008-12-01

    We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features. PMID:18845569

  19. A conformational study of the binding of a high mobility group protein with chromatin.

    PubMed

    Sasi, R; Hüvös, 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 demonstrated that a condensation of chromatin structure occurred upon HMG 17 binding at low ionic strength (1 mM Na phosphate, 0.25 mM EDTA, pH 7.0). Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. In general, a shift in the temperature of maximum dh/dT for each transition (Tm) was observed for all transitions upon HMG 17 binding. DNA melting in the first transition, originating from linker regions of whole chromatin, was nearly totally depleted and was distributed mainly into the highest melting transition. The same trends were also observed in H1-H5-depleted chromatin. 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 (50 mM NaCl, 1 mM Na phosphate, 0.25 mM EDTA, pH 7.0) with whole chromatin and H1-H5-depleted chromatin was found to be different but a decrease in [theta] values was found in both chromatins. 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. PMID:6214552

  20. Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling.

    PubMed

    Illi, Barbara; Dello Russo, Claudio; Colussi, Claudia; Rosati, Jessica; Pallaoro, Michele; Spallotta, Francesco; Rotili, Dante; Valente, Sergio; Ragone, Gianluca; Martelli, Fabio; Biglioli, Paolo; Steinkuhler, Christian; Gallinari, Paola; Mai, Antonello; Capogrossi, Maurizio C; Gaetano, Carlo

    2008-01-01

    Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation. PMID:17975112

  1. Mammalian sperm chromatin as a model for chromatin function in DNA degradation and DNA replication.

    PubMed

    Ortega, Michael A; Sil, Payel; Ward, W Steven

    2011-02-01

    Reproductive biology is considered a specialty field, however, an argument can be made that it is instead generally applicable to many fields of biology. The one-cell embryo is presented here as a model system for the study of eukaryotic DNA replication, apoptotic DNA degradation, and signaling mechanisms between the cytoplasm and nucleus. Two unique aspects of this system combine to make it particularly useful for the study of chromatin function. First, the evolutionary pressure that lead to the extreme condensation of mammalian sperm DNA resulted in a cell with virtually inert chromatin, no DNA replication or transcription ongoing in the sperm cell, and all of the cells in a G(0) state. This chromatin is suddenly transformed into actively transcribing and replicating DNA upon fertilization. Therefore, the sperm chromatin is poised to become active but does not yet possess sufficient components present in somatic chromatin structure for all these processes. The second unique aspect of this system is that the one cell embryo houses two distinct nuclei, termed pronuclei, through the first round of DNA synthesis. This means the sperm cell can be experimentally manipulated to test the affects of the various treatments on the biological functions of interest. Experimental manipulations of the system have already revealed a certain level of plasticity in the coordination of both the timing of DNA synthesis in the two pronuclei and in the response to cellular signals by each pronucleus involved with the progression through the G1/S checkpoint, including the degradation of DNA in the paternal pronucleus. The fact that two nuclei in the same cytoplasm can undergo different responses infers a level of autonomy in the nuclear control of the cell cycle. Thus, the features of mammalian fertilization can provide unique insights for the normal biology of the cell cycle in somatic cells. PMID:21204750

  2. Quantifying chromatin-associated interactions: the HI-FI system.

    PubMed

    Winkler, Duane D; Luger, Karolin; Hieb, Aaron R

    2012-01-01

    Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific factors recognize and act upon the various physical states of chromatin to modulate DNA accessibility. The ability to quantitatively characterize these interactions in vitro can provide valuable insight into the mechanisms that dictate chromatin architecture. Here, we describe in detail fluorescence methodologies for quantifying the thermodynamic principles that guide interactions between nucleosomal arrays, mononucleosomes, or nucleosome components and chromatin-associated factors through application of the HI-FI (High-throughput Interactions by Fluorescence Intensity) system. These measurements utilize fluorescence (de)quenching and FRET assays performed in 384-well microplates, making the assays suitable for high-throughput characterization of interactions at low concentrations. Further, this system can be used to determine the stoichiometric composition of complexes and specific sites of interaction. After quantification on a plate reader or similar instrument, the solution-based assays can be directly transferred to native gels for visualization of interaction(s). We also highlight procedural details on the efficient attachment of fluorescent dyes to histones and DNA. In all, the HI-FI system of assays can be used to elucidate mechanistic details of how specific chromatin-associated factors function at the molecular level. PMID:22910210

  3. Cas9 Functionally Opens Chromatin.

    PubMed

    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

  4. 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

  5. Conservation laws and dark-soliton solutions of an integrable higher-order nonlinear Schrödinger equation for a density-modulated quantum condensate

    NASA Astrophysics Data System (ADS)

    Liu, De-Yin; Tian, Bo; Sun, Wen-Rong; Wang, Yun-Po

    2015-04-01

    In this paper, an integrable higher-order nonlinear Schrödinger equation for a density-modulated quantum condensate is investigated. Based on the Ablowitz-Kaup-Newell-Segur system, an infinite number of conservation laws are obtained. Introducing an auxiliary function, we derive the bilinear forms and construct the dark-soliton solutions with the help of the Hirota method and symbolic computation. Dark one, two, and three solitons are analyzed graphically. Via asymptotic analysis, interactions between the two dark solitons are proved to be elastic. We see that the coefficients in the equation only affect the soliton velocity. We analyze the linear stability of the plane wave solutions in the presence of a small perturbation.

  6. Mechanism of chromatin remodeling.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2010-02-23

    Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling. PMID:20142505

  7. 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.

  8. Effects of cryostorage on human sperm chromatin integrity.

    PubMed

    Fortunato, Adriana; Leo, Rita; Liguori, Francesca

    2013-11-01

    The integrity of sperm chromatin structure has proven to be of great importance for human fertility. In this study, we investigated whether sperm cryopreservation has an effect on nuclear DNA tertiary structure, (i.e. condensation), measured by aniline blue staining, in 103 male patients who required consultation for hypo-fertility. Sperm DNA damage was significantly higher in patients showing oligospermia and severe morphological abnormalities than in native sperm populations. Furthermore we observed that chromatin decondensation was related to the cryostorage technique and to the duration of storage. This increase in decondensation was highly significant (P < 0.01) immediately after cryopreservation and from 90 days of cryostorage onwards. The possible mechanisms involved in sperm chromatin cryoinjury and the need to incorporate new methods for testing sperm nuclear structure alteration into the routine spermiogram are discussed. PMID:22398023

  9. 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. PMID:26868054

  10. [Cytophotometric analysis of the chromatin structural conformity in interphase nuclei detected in UV light and by gallocyanine staining].

    PubMed

    Zhukotskiĭ, A V; Shchegolev, A I; Butusova, N N; Nemirovskiĭ, L E; Kogan, E M

    1985-06-01

    Geometric and optical parameters of chromatin of hepatocyte nuclei have been examined before (UV, lambda = 265 nm) and after gallocyanine staining. Quantitative parameters of the chromatin structure in the same nuclei measured in situ by a scanning microscope-photometer (step size 0.125 micron) before and after staining were equal. Tinctorial properties of chromatin granules (condensed part of the nuclear material) and its diffuse part were different. It is suggested that the difference between granules and the nongranular part of chromatin is not only of optical but also of chemical nature. PMID:2410060

  11. A WD-repeat protein stabilizes ORC binding to chromatin.

    PubMed

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

    2010-10-01

    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

  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. Ultrastructure of bovine sperm chromatin.

    PubMed

    Filho, Romualdo Morandi; Beletti, Marcelo Emilio; de Oliveira, Fabio

    2015-12-01

    Mammalian semen chromatin comprises DNA, protamine, and, at lower levels, other proteins. This constitution confers intense compaction to the chromatin, helping to protect the DNA and causing the head of the sperm to be very small, facilitating the safe transport of its genetic contents. It is known that changes in the sperm chromatin compaction lead to fertility problems in bulls, justifying studies of this structure. Although there are theoretical models of sperm chromatin because of its high compaction, there is no morphological evidence of such models. The aim of this study was to demonstrate the ultrastructure of bovine sperm chromatin in an attempt to corroborate the theoretical chromatin models existing today. The isolated bull sperm heads had their chromatin partially unpacked by chemical treatment using sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and were then embedded in Epon resin. Using an ultramicrotome, ultrathin sections were obtained, which were contrasted with uranyl acetate and lead citrate, and then viewed under transmission electron microscopy. The methodology used allowed the visualization of toroidal structures interconnected by a filamentous nuclear matrix, which is entirely consistent with the most current theoretical models. PMID:26515508

  14. Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

    PubMed

    Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z; Wickrema, Amittha; Yang, Jing; Ji, Peng

    2016-03-01

    Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice. PMID:26954545

  15. Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

    PubMed

    Lee, Sung Won; Park, Hyun Jung; Jeon, Sung Ho; Lee, Changjin; Seong, Rho Hyun; Park, Se-Ho; Hong, Seokmann

    2015-01-01

    Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization

  16. Chromatin, epigenetics and stem cells.

    PubMed

    Roloff, Tim C; Nuber, Ulrike A

    2005-03-01

    Epigenetics is a term that has changed its meaning with the increasing biological knowledge on developmental processes. However, its current application to stem cell biology is often imprecise and is conceptually problematic. This article addresses two different subjects, the definition of epigenetics and chromatin states of stem and differentiated cells. We describe mechanisms that regulate chromatin changes and provide an overview of chromatin states of stem and differentiated cells. Moreover, a modification of the current epigenetics definition is proposed that is not restricted by the heritability of gene expression throughout cell divisions and excludes translational gene expression control. PMID:15819395

  17. Chromatin organization: form to function.

    PubMed

    de Graaf, Carolyn A; van Steensel, Bas

    2013-04-01

    Recent developments in technology have made it possible to create high resolution genome-wide maps of histone marks, DNA binding proteins and physical interactions along genomic regions. Chromatin features are found together in different combinations, dividing the genome up into domains with distinct functional properties. Microscopy and chromatin conformation capture techniques have shown that the 3D structure of chromosomes is constrained by nuclear features and functional links between different parts of chromatin. These results provide insights about the 3D and domain organization of the genome and their connection to gene regulation and other nuclear functions. PMID:23274160

  18. CONDENSATION CAN

    DOEpatents

    Booth, E.T. Jr.; Pontius, R.B.; Jacobsohn, B.A.; Slade, C.B.

    1962-03-01

    An apparatus is designed for condensing a vapor to a solid at relatively low back pressures. The apparatus comprises a closed condensing chamber, a vapor inlet tube extending to the central region of the chamber, a co-axial tubular shield surrounding the inlet tube, means for heating the inlet tube at a point outside the condensing chamber, and means for refrigeratirg the said chamber. (AEC)

  19. Painting a Clearer Picture of Chromatin.

    PubMed

    Finn, Elizabeth H; Misteli, Tom; Shachar, Sigal

    2016-02-22

    Elucidating chromatin's 3D shape is critical to understanding its function, but the fine structure of chromatin domains remains poorly resolved. In a recent report in Nature, Boettiger et al. (2016) visualize chromatin in super-resolution, gaining unprecedented insight into chromatin architecture. PMID:26906730

  20. Non-Coding RNA: Sequence-Specific Guide for Chromatin Modification and DNA Damage Signaling

    PubMed Central

    Francia, Sofia

    2015-01-01

    Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair. PMID:26617633

  1. 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

  2. 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. PMID:19799187

  3. Critical electrolyte concentration of silk gland chromatin of the sugarcane borer Diatraea saccharalis, induced using agrochemicals.

    PubMed

    Santos, S A; Fermino, F; Moreira, B M T; Araujo, K F; Falco, J R P; Ruvolo-Takasusuki, M C C

    2014-01-01

    The sugarcane borer Diatraea saccharalis is widely known as the main pest of sugarcane crop, causing increased damage to the entire fields. Measures to control this pest involve the use of chemicals and biological control with Cotesia flavipes wasps. In this study, we evaluated the insecticides fipronil (Frontline; 0.0025%), malathion (Malatol Bio Carb; 0.4%), cipermetrina (Galgotrin; 10%), and neem oil (Natuneem; 100%) and the herbicide nicosulfuron (Sanson 40 SC; 100%) in the posterior region silk glands of 3rd- and 5th-instar D. saccharalis by studying the variation in the critical electrolyte concentration (CEC). Observations of 3rd-instar larvae indicated that malathion, cipermetrina, and neem oil induced increased chromatin condensation that may consequently disable genes. Tests with fipronil showed no alteration in chromatin condensation. With the use of nicosulfuron, there was chromatin and probable gene decompaction. In the 5th-instar larvae, the larval CEC values indicated that malathion and neem oil induced increased chromatin condensation. The CEC values for 5th-instar larvae using cipermetrina, fipronil, and nicosulfuron indicated chromatin unpacking. These observations led us to conclude that the quantity of the pesticide does not affect the mortality of these pests, can change the conformation of complexes of DNA, RNA, and protein from the posterior region of silk gland cells of D. saccharalis, activating or repressing the expression of genes related to the defense mechanism of the insect and contributing to the selection and survival of resistant individuals. PMID:25299111

  4. Polariton Condensation in Dynamic Acoustic Lattices

    NASA Astrophysics Data System (ADS)

    Cerda-Méndez, E. A.; Krizhanovskii, D. N.; Wouters, M.; Bradley, R.; Biermann, K.; Guda, K.; Hey, R.; Santos, P. V.; Sarkar, D.; Skolnick, M. S.

    2010-09-01

    We demonstrate that the tunable potential introduced by a surface acoustic wave on a homogeneous polariton condensate leads to fragmentation of the condensate into an array of wires which move with the acoustic velocity. Reduction of the spatial coherence of the condensate emission along the surface acoustic wave direction is attributed to the suppression of coupling between the spatially modulated condensates. Interparticle interactions observed at high polariton densities screen the acoustic potential, partially reversing its effect on spatial coherence.

  5. Genome accessibility is widely preserved and locally modulated during mitosis

    PubMed Central

    Hsiung, Chris C.-S.; Morrissey, Christapher S.; Udugama, Maheshi; Frank, Christopher L.; Keller, Cheryl A.; Baek, Songjoon; Giardine, Belinda; Crawford, Gregory E.; Sung, Myong-Hee; Hardison, Ross C.

    2015-01-01

    Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. PMID:25373146

  6. Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing

    PubMed Central

    Tashiro, Sanki; Handa, Tetsuya; Matsuda, Atsushi; Ban, Takuto; Takigawa, Toru; Miyasato, Kazumi; Ishii, Kojiro; Kugou, Kazuto; Ohta, Kunihiro; Hiraoka, Yasushi; Masukata, Hisao; Kanoh, Junko

    2016-01-01

    A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body ‘knob'. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing. PMID:26804021

  7. Regulation of chromatin structure by poly(ADP-ribosyl)ation

    PubMed Central

    Beneke, Sascha

    2012-01-01

    The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose) has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose), the zinc-finger protein poly(ADP-ribose) polymerase-1 (PARP1), was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor. PMID:22969794

  8. The physical size of transcription factors is key to transcriptional regulation in chromatin domains

    NASA Astrophysics Data System (ADS)

    Maeshima, Kazuhiro; Kaizu, Kazunari; Tamura, Sachiko; Nozaki, Tadasu; Kokubo, Tetsuro; Takahashi, Koichi

    2015-02-01

    Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (˜50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a ‘buoy’ to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

  9. The physical size of transcription factors is key to transcriptional regulation in chromatin domains.

    PubMed

    Maeshima, Kazuhiro; Kaizu, Kazunari; Tamura, Sachiko; Nozaki, Tadasu; Kokubo, Tetsuro; Takahashi, Koichi

    2015-02-18

    Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (∼50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a 'buoy' to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination. PMID:25563431

  10. Chromatin Decondensation and Nuclear Softening Accompany Nanog Downregulation in Embryonic Stem Cells

    PubMed Central

    Chalut, Kevin J.; Höpfler, Markus; Lautenschläger, Franziska; Boyde, Lars; Chan, Chii Jou; Ekpenyong, Andrew; Martinez-Arias, Alfonso; Guck, Jochen

    2012-01-01

    The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself—even prior to differentiation—is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development. PMID:23200040

  11. Global quantitative modeling of chromatin factor interactions.

    PubMed

    Zhou, Jian; Troyanskaya, Olga G

    2014-03-01

    Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the "chromatin codes") remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles--we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

  12. 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

  13. Structural and functional genome analysis using extended chromatin

    SciTech Connect

    Heaf, T.; Ward, D.C.

    1994-09-01

    Highly extended linear chromatin fibers (ECFs) produced by detergent and high-salt lysis and stretching of nuclear chromatin across the surface of a glass slide can by hybridized over physical distances of at least several Mb. This allows long-range FISH analysis of the human genome with excellent DNA resolution (<10 kb/{mu}m). The insertion of Alu elements which are more than 50-fold underrepresented in centromeres can be seen within and near long tandem arrays of alpha-satellite DNA. Long tracts of trinucleotide repeats, i.e. (CCA){sub n}, can be localized within larger genomic regions. The combined application of BrdU incorporation and ECFs allows one to study the spatio-temporal distribution of DNA replication sites in finer detail. DNA synthesis occurs at multiple discrete sites within Mb arrays of alpha-satellite. Replicating DNA is tightly associated with the nuclear matrix and highly resistant to stretching out, while ECFs containing newly replicated DNA are easily released. Asynchrony in replication timing is accompanied by differences in condensation of homologous DNA segments. Extended chromatin reveals differential packaging of active and inactive DNA. Upon transcriptional inactivation by AMD, the normally compact rRNA genes become much more susceptible to decondensation procedures. By extending the chromatin from pachytene spermatocytes, meiotic pairing and genetic exchange between homologs can be visualized directly. Histone depletion by high salt and detergent produces loop chromatin surrounding the nuclear matrix in a halo-like fashion. DNA halos can be used to map nuclear matrix attachment sites in somatic cells and in mature sperm. Alpha-satellite containing DNA loops appear to be attached to the sperm-cell matrix by CENP-B boxes, short 17 bp sequences found in a subset of alpha satellite monomers. Sperm telomeres almost always appear as hybridization doublets, suggesting the presence of already replicated chromosome ends.

  14. Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

    PubMed Central

    Panday, Arvind; Xiao, LiJuan; Grove, Anne

    2015-01-01

    DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair. PMID:25979266

  15. Cytomixis doesn't induce obvious changes in chromatin modifications and programmed cell death in tobacco male meiocytes.

    PubMed

    Mursalimov, Sergey; Permyakova, Natalya; Deineko, Elena; Houben, Andreas; Demidov, Dmitri

    2015-01-01

    Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin. PMID:26528310

  16. Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

    Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing. PMID:16914722

  17. Cytomixis doesn’t induce obvious changes in chromatin modifications and programmed cell death in tobacco male meiocytes

    PubMed Central

    Mursalimov, Sergey; Permyakova, Natalya; Deineko, Elena; Houben, Andreas; Demidov, Dmitri

    2015-01-01

    Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin. PMID:26528310

  18. Condensation polyimides

    NASA Technical Reports Server (NTRS)

    Hergenrother, P. M.

    1989-01-01

    Polyimides belong to a class of polymers known as polyheterocyclics. Unlike most other high temperature polymers, polyimides can be prepared from a variety of inexpensive monomers by several synthetic routes. The glass transition and crystalline melt temperature, thermooxidative stability, toughness, dielectric constant, coefficient of thermal expansion, chemical stability, mechanical performance, etc. of polyimides can be controlled within certain boundaries. This versatility has permitted the development of various forms of polyimides. These include adhesives, composite matrices, coatings, films, moldings, fibers, foams and membranes. Polyimides are synthesized through both condensation (step-polymerization) and addition (chain growth polymerization) routes. The precursor materials used in addition polyimides or imide oligomers are prepared by condensation method. High molecular weight polyimide made via polycondensation or step-growth polymerization is studied. The various synthetic routes to condensation polyimides, structure/property relationships of condensation polyimides and composite properties of condensation polyimides are all studied. The focus is on the synthesis and chemical structure/property relationships of polyimides with particular emphasis on materials for composite application.

  19. Implementation of non-condensable gases condensation suppression model into the WCOBRA/TRAC-TF2 LOCA safety evaluation code

    SciTech Connect

    Liao, J.; Cao, L.; Ohkawa, K.; Frepoli, C.

    2012-07-01

    The non-condensable gases condensation suppression model is important for a realistic LOCA safety analysis code. A condensation suppression model for direct contact condensation was previously developed by Westinghouse using first principles. The model is believed to be an accurate description of the direct contact condensation process in the presence of non-condensable gases. The Westinghouse condensation suppression model is further revised by applying a more physical model. The revised condensation suppression model is thus implemented into the WCOBRA/TRAC-TF2 LOCA safety evaluation code for both 3-D module (COBRA-TF) and 1-D module (TRAC-PF1). Parametric study using the revised Westinghouse condensation suppression model is conducted. Additionally, the performance of non-condensable gases condensation suppression model is examined in the ACHILLES (ISP-25) separate effects test and LOFT L2-5 (ISP-13) integral effects test. (authors)

  20. Chromatin Structure Regulates Gene Conversion

    PubMed Central

    Cummings, W. Jason; Yabuki, Munehisa; Ordinario, Ellen C; Bednarski, David W; Quay, Simon; Maizels, Nancy

    2007-01-01

    Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences. PMID:17880262

  1. Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

    PubMed Central

    Chesterton, C. James; Coupar, Barbara E. H.; Butterworth, Peter H. W.

    1974-01-01

    The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA. PMID:4464858

  2. Discovering Cooperative Relationships of Chromatin Modifications in Human T Cells Based on a Proposed Closeness Measure

    PubMed Central

    Liu, Hongbo; Wu, Xueting; Zhu, Jiang; Su, Jianzhong; Wang, Fang; Cui, Ying; Zhang, Yan

    2010-01-01

    Background Eukaryotic transcription is accompanied by combinatorial chromatin modifications that serve as functional epigenetic markers. Composition of chromatin modifications specifies histone codes that regulate the associated gene. Discovering novel chromatin regulatory relationships are of general interest. Methodology/Principal Findings Based on the premise that the interaction of chromatin modifications is hypothesized to influence CpG methylation, we present a closeness measure to characterize the regulatory interactions of epigenomic features. The closeness measure is applied to genome-wide CpG methylation and histone modification datasets in human CD4+T cells to select a subset of potential features. To uncover epigenomic and genomic patterns, CpG loci are clustered into nine modules associated with distinct chromatin and genomic signatures based on terms of biological function. We then performed Bayesian network inference to uncover inherent regulatory relationships from the feature selected closeness measure profile and all nine module-specific profiles respectively. The global and module-specific network exhibits topological proximity and modularity. We found that the regulatory patterns of chromatin modifications differ significantly across modules and that distinct patterns are related to specific transcriptional levels and biological function. DNA methylation and genomic features are found to have little regulatory function. The regulatory relationships were partly validated by literature reviews. We also used partial correlation analysis in other cells to verify novel regulatory relationships. Conclusions/Significance The interactions among chromatin modifications and genomic elements characterized by a closeness measure help elucidate cooperative patterns of chromatin modification in transcriptional regulation and help decipher complex histone codes. PMID:21151929

  3. Regulation of DNA transposition by CpG methylation and chromatin structure in human cells

    PubMed Central

    2013-01-01

    Background The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. Results In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. Conclusions We have shown that DNA CpG methylation

  4. 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

  5. In vitro chromatin templates to study nucleotide excision repair.

    PubMed

    Liu, Xiaoqi

    2015-12-01

    In eukaryotic cells, DNA associates with histones and exists in the form of a chromatin hierarchy. Thus, it is generally believed that many eukaryotic cellular DNA processing events such as replication, transcription, recombination and DNA repair are influenced by the packaging of DNA into chromatin. This mini-review covers the current knowledge of DNA damage and repair in chromatin based on in vitro studies. Specifically, nucleosome assembly affects DNA damage formation in both random sequences and sequences with strong nucleosome-positioning signals such as 5S rDNA. At least three systems have been used to analyze the effect of nucleosome folding on nucleotide excision repair (NER) in vitro: (a) human cell extracts that have to rely on labeling of repair synthesis to monitor DNA repair, due to very low repair efficacy; (b) Xenopus oocyte nuclear extracts, that have very robust DNA repair efficacy, have been utilized to follow direct removal of DNA damage; (c) six purified human DNA repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1) that have been used to reconstitute excision repair in vitro. In general, the results have shown that nucleosome folding inhibits NER and, therefore, its activity must be enhanced by chromatin remodeling factors like SWI/SNF. In addition, binding of transcription factors such as TFIIIA to the 5S rDNA promoter also modulates NER efficacy. PMID:26531320

  6. 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

  7. A chromatin perspective of adipogenesis

    PubMed Central

    Musri, Melina M; Gomis, Ramon

    2010-01-01

    The transcriptional cascade governing adipogenesis has been thoroughly examined throughout the years. Transcription factors PPARγ and C/EBPα are universally recognized as the master regulators of adipocyte differentiation and together they direct the establishment of the gene expression pattern of mature adipose cells. However, this familiar landscape has been considerably broadened in recent years by the identification of novel factors that participate in the regulation of adipogenesis, either favoring or inhibiting it, through their effects on chromatin. Epigenetic signals and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism. In this review we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade. PMID:20592861

  8. Residual chromatin breaks as biodosimetry for cell killing by carbon ions.

    PubMed

    Suzuki, M; Kase, Y; Nakano, T; Kanai, T; Ando, K

    1998-01-01

    We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET = 13 keV/micrometer, 76 keV/micrometer) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour post-irradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/micrometer beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy. PMID:11542410

  9. Residual chromatin breaks as biodosimetry for cell killing by carbon ions

    NASA Astrophysics Data System (ADS)

    Suzuki, M.; Kase, Y.; Nakano, T.; Kanai, T.; Ando, K.

    1998-11-01

    We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET = 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour post-irradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.

  10. Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

    SciTech Connect

    Tone, Shigenobu Sugimoto, Kenji; Tanda, Kazue; Suda, Taiji; Uehira, Kenzo; Kanouchi, Hiroaki; Samejima, Kumiko; Minatogawa, Yohsuke; Earnshaw, William C.

    2007-10-01

    During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.

  11. Estimation of thermoelastic deformations in the near-surface layers of condensed media upon absorption of time-modulated laser radiation

    NASA Astrophysics Data System (ADS)

    Muratikov, K. L.

    2015-10-01

    Nonstationary thermoelastic deformation in the near-surface layers of condensed media are determined in the quasi-static approximation. The deformations are induced by the absorbed laser radiation. A 3D model is used to derive analytical expressions for normal and radial components of the deformation vector of the surface.

  12. Imaging of DNA/Nanosphere Condensates

    NASA Astrophysics Data System (ADS)

    Krishnan, R.

    2005-03-01

    DNA forms condensates in a variety of environments. In chromatin, DNA is condensed around 10-nm-diameter, positively-charged histone complexes. To model chromatin formation in cells, lambda-phage (16 microns long) and herring sperm (0.03 to1 micron) DNAs were mixed with polystyrene nanospheres of diameter 40nm and 930nm containing 1.8x10^4 and 2.6x10^8 positive surface charges, respectively, to form condensates. Sphere concentrations were 1-2 times the isoelectric concentration. Condensation vs time was imaged at various concentrations, pH's, viscosities, and ionic strengths. Bright-field and fluorescence (YOYO-1 dye bound to DNA) images were recorded. In general HS DNA aggregate size increased with time. Except in 0.5-0.8 M KCl, herring sperm DNA formed one huge aggregate (100's of microns) and depleted other areas, both in 10% and 20% glycerol. Phage DNA samples rapidly formed longer, fiber-like aggregates. Within 2 hours it formed ordered structures and in most samples, empty, apparently depleted regions were found in the viewing area. Shapes of the phage-DNA aggregates in 20% glycerol, in contrast, formed small clumps like HS DNA.

  13. Correlation among DNA Linker Length, Linker Histone Concentration, and Histone Tails in Chromatin.

    PubMed

    Luque, Antoni; Ozer, Gungor; Schlick, Tamar

    2016-06-01

    Eukaryotic cells condense their genetic material in the nucleus in the form of chromatin, a macromolecular complex made of DNA and multiple proteins. The structure of chromatin is intimately connected to the regulation of all eukaryotic organisms, from amoebas to humans, but its organization remains largely unknown. The nucleosome repeat length (NRL) and the concentration of linker histones (ρLH) are two structural parameters that vary among cell types and cell cycles; the NRL is the number of DNA basepairs wound around each nucleosome core plus the number of basepairs linking successive nucleosomes. Recent studies have found a linear empirical relationship between the variation of these two properties for different cells, but its underlying mechanism remains elusive. Here we apply our established mesoscale chromatin model to explore the mechanisms responsible for this relationship, by investigating chromatin fibers as a function of NRL and ρLH combinations. We find that a threshold of linker histone concentration triggers the compaction of chromatin into well-formed 30-nm fibers; this critical value increases linearly with NRL, except for long NRLs, where the fibers remain disorganized. Remarkably, the interaction patterns between core histone tails and chromatin elements are highly sensitive to the NRL and ρLH combination, suggesting a molecular mechanism that could have a key role in regulating the structural state of the fibers in the cell. An estimate of the minimized work and volume associated with storage of chromatin fibers in the nucleus further suggests factors that could spontaneously regulate the NRL as a function of linker histone concentration. Both the tail interaction map and DNA packing considerations support the empirical NRL/ρLH relationship and offer a framework to interpret experiments for different chromatin conditions in the cell. PMID:27276249

  14. Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

    PubMed

    Tiano, L; Chessa, M G; Carrara, S; Tagliafierro, G; Delmonte Corrado, M U

    1999-01-01

    The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal. PMID:10439214

  15. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

    PubMed Central

    Ulianov, Sergey V.; Khrameeva, Ekaterina E.; Gavrilov, Alexey A.; Flyamer, Ilya M.; Kos, Pavel; Mikhaleva, Elena A.; Penin, Aleksey A.; Logacheva, Maria D.; Imakaev, Maxim V.; Chertovich, Alexander; Gelfand, Mikhail S.; Shevelyov, Yuri Y.; Razin, Sergey V.

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)+ RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin. PMID:26518482

  16. Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

    PubMed Central

    Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

    2016-01-01

    ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

  17. Identification of lamin B-regulated chromatin regions based on chromatin landscapes.

    PubMed

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-07-15

    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

  18. 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.

  19. Condensed-phase thermal decomposition of TATB investigated by atomic force microscopy (AFM) and simultaneous thermogravimetric modulated beam mass spectrometry (STMBMS)

    SciTech Connect

    Land, T.A.; Siekhaus, W.J.; Foltz, M.F.; Behrens, R. Jr.

    1993-05-01

    A combination of techniques has been used to investigate the condensed-phase thermal decomposition of TATB. STMBMS has been used to identify the thermal decomposition products and their temporal correlation`s. These experiments have shown that the condensed-phase decomposition proceeds through several autocatalytic pathways. Both low and high molecular weight decomposition products have been identified. Mono-, di- and tri-furazans products have been identified and, their temporal behaviors are consistent with a stepwise loss of water. AFM has been used to correlate the decomposition chemistry with morphological changes occurring as a function of heating. Patches of small 25-140 nm round holes were observed throughout the lattice of TATB crystals that were heated briefly to 300C. It is likely that these holes show where decomposition reactions have started. Evidence of decomposition products have been seen in TATB that has been held at 250C for one hour.

  20. 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

  1. Chromatin Structure and Dynamics in Hot Environments: Architectural Proteins and DNA Topoisomerases of Thermophilic Archaea

    PubMed Central

    Visone, Valeria; Vettone, Antonella; Serpe, Mario; Valenti, Anna; Perugino, Giuseppe; Rossi, Mosè; Ciaramella, Maria

    2014-01-01

    In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity. PMID:25257534

  2. Open chromatin in pluripotency and reprogramming

    PubMed Central

    Meshorer, Eran; Ramalho-Santos, Miguel

    2013-01-01

    Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique from any other stem cell in that they can give rise to all cell types of the body. Recent findings indicate that a particularly open chromatin state contributes to maintenance of pluripotency. Two emerging principles are that: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer. PMID:21179060

  3. Single cell correlation fractal dimension of chromatin

    PubMed Central

    Récamier, Vincent; Izeddin, Ignacio; Bosanac, Lana; Dahan, Maxime; Proux, Florence; Darzacq, Xavier

    2014-01-01

    Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state. PMID:24637833

  4. Epigenetic Regulation by ATP-Dependent Chromatin-Remodeling Enzymes: SNF-ing Out Crosstalk.

    PubMed

    Runge, John S; Raab, Jesse R; Magnuson, Terry

    2016-01-01

    Cells utilize precise mechanisms to access genomic DNA with spatiotemporal accuracy. ATP-dependent chromatin-remodeling enzymes (also known simply as "remodelers") comprise a specialized class of enzymes that is intimately involved in genomic organization and accessibility. Remodelers selectively position nucleosomes to either alleviate chromatin compaction or achieve genomic condensation locally, based on a multitude of cellular signals. By dictating nucleosome position, remodelers control local euchromatic and heterochromatic states. These activities govern the accessibility of regulatory regions like promoters and enhancers to transcription factors, RNA polymerases, and coactivators or -repressors. As studies unravel the complexities of epigenetic topography, evidence points to a chromatin-based interactome where regulators interact competitively, cooperatively, and/or codependently through physical and functional means. These types of interactions, or crosstalk, between remodelers raise important questions for tissue development. Here, we briefly review the evidence for remodeler interactions and argue for additional studies examining crosstalk. PMID:26969969

  5. 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

  6. Fourier transform infrared spectroscopic analysis of sperm chromatin structure and DNA stability.

    PubMed

    Oldenhof, H; Schütze, S; Wolkers, W F; Sieme, H

    2016-05-01

    Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and packing. Spectroscopic studies were corroborated with flow cytometric analyses using the DNA-intercalating fluorescent dye acridine orange. Decreased fertility of individuals correlated with increased abnormal sperm morphology and decreased stability toward induced DNA damage. Treatment with the disulfide reducing agent dithiothreitol resulted in increased sperm chromatin decondensation and DNA accessibility, similar as found for less mature epididymal spermatozoa. In situ infrared spectroscopic analysis revealed that characteristic bands arising from the DNA backbone (ν1230, ν1086, ν1051 cm(-1) ) changed in response to induced oxidative damage, water removal, and decondensation. This coincided with changes in the amide-I region (intensity at ν1620 vs. ν1640 cm(-1) ) denoting concomitant changes in protein secondary structure. Reduction in protein disulfide bonds resulted in a decreased value of the asymmetric to symmetric phosphate band intensity (ν1230/ν1086 cm(-1) ), suggesting that this band ratio is sensitive for the degree of chromatin condensation. Moreover, when analyzing spermatozoa from different individuals, it was found that the asymmetric/symmetric phosphate band ratio negatively correlated with the percentage of morphologically abnormal spermatozoa. PMID:26916383

  7. Structure, Assembly and Reading of Centromeric Chromatin

    PubMed Central

    Maddox, Paul S; Corbett, Kevin D; Desai, Arshad

    2011-01-01

    Centromeres are epigenetically defined chromatin domains marked by the presence of the histone H3 variant CENP-A. Here we review recent structural and biochemical work on CENP-A, and advances in understanding the mechanisms that propagate and read centromeric chromatin domains. PMID:22178421

  8. Chromatin Remodelers: From Function to Dysfunction

    PubMed Central

    Längst, Gernot; Manelyte, Laura

    2015-01-01

    Chromatin remodelers are key players in the regulation of chromatin accessibility and nucleosome positioning on the eukaryotic DNA, thereby essential for all DNA dependent biological processes. Thus, it is not surprising that upon of deregulation of those molecular machines healthy cells can turn into cancerous cells. Even though the remodeling enzymes are very abundant and a multitude of different enzymes and chromatin remodeling complexes exist in the cell, the particular remodeling complex with its specific nucleosome positioning features must be at the right place at the right time in order to ensure the proper regulation of the DNA dependent processes. To achieve this, chromatin remodeling complexes harbor protein domains that specifically read chromatin targeting signals, such as histone modifications, DNA sequence/structure, non-coding RNAs, histone variants or DNA bound interacting proteins. Recent studies reveal the interaction between non-coding RNAs and chromatin remodeling complexes showing importance of RNA in remodeling enzyme targeting, scaffolding and regulation. In this review, we summarize current understanding of chromatin remodeling enzyme targeting to chromatin and their role in cancer development. PMID:26075616

  9. Single Chromatin Fiber Stretching Reveals Physically Distinct Populations of Disassembly Events

    PubMed Central

    Pope, L. H.; Bennink, M. L.; van Leijenhorst-Groener, K. A.; Nikova, D.; Greve, J.; Marko, J. F.

    2005-01-01

    Eukaryotic DNA is packaged into the cell nucleus as a nucleoprotein complex, chromatin. Despite this condensed state, access to the DNA sequence must occur during gene expression and other essential genetic events. Here we employ optical tweezers stretching of reconstituted chromatin fibers to investigate the release of DNA from its protein-bound structure. Analysis of fiber length increase per unbinding event revealed discrete values of ∼30 and ∼60 nm. Furthermore, a loading rate analysis of the disruption forces revealed three individual energy barriers. The heights of these barriers were found to be ∼20 kBT, ∼25 kBT, and ∼28 kBT. For subsequent stretches of the fiber it was found that events corresponding to the ∼28 kBT energy barrier were significantly reduced. No correlation between energy barrier crossed and DNA length release was found. These studies clearly demonstrate that optical tweezers stretching of chromatin provides insight into the energetic penalties imposed by chromatin structure. Furthermore these studies reveal possible pathways via which chromatin may be disrupted during genetic code access. PMID:15695630

  10. Organization of spacer DNA in chromatin.

    PubMed Central

    Lohr, D; Van Holde, K E

    1979-01-01

    Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature. Images PMID:392519

  11. 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

  12. RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking

    PubMed Central

    Choi, Inchan; Won, Kyoung-Jae; Fan, Hua-Ying

    2014-01-01

    Mechanisms that maintain transcriptional memory through cell division are important to maintain cell identity, and sequence-specific transcription factors that remain associated with mitotic chromatin are emerging as key players in transcriptional memory propagation. Here, we show that the major transcriptional effector of Notch signaling, RBPJ, is retained on mitotic chromatin, and that this mitotic chromatin association is mediated through the direct association of RBPJ with DNA. We further demonstrate that RBPJ binds directly to nucleosomal DNA in vitro, with a preference for sites close to the entry/exit position of the nucleosomal DNA. Genome-wide analysis in the murine embryonal-carcinoma cell line F9 revealed that roughly 60% of the sites occupied by RBPJ in asynchronous cells were also occupied in mitotic cells. Among them, we found that a fraction of RBPJ occupancy sites shifted between interphase and mitosis, suggesting that RBPJ can be retained on mitotic chromatin by sliding on DNA rather than disengaging from chromatin during mitotic chromatin condensation. We propose that RBPJ can function as a mitotic bookmark, marking genes for efficient transcriptional activation or repression upon mitotic exit. Strikingly, we found that sites of RBPJ occupancy were enriched for CTCF-binding motifs in addition to RBPJ-binding motifs, and that RBPJ and CTCF interact. Given that CTCF regulates transcription and bridges long-range chromatin interactions, our results raise the intriguing hypothesis that by collaborating with CTCF, RBPJ may participate in establishing chromatin domains and/or long-range chromatin interactions that could be propagated through cell division to maintain gene expression programs. PMID:24603501

  13. Effete, an E2 ubiquitin-conjugating enzyme with multiple roles in Drosophila development and chromatin organization

    PubMed Central

    Cipressa, Francesca; Cenci, Giovanni

    2013-01-01

    The Drosophila effete gene encodes an extremely conserved class I E2 ubiquitin-conjugating enzyme. Growing evidence indicates that Eff is involved in many cellular processes including eye development, maintenance of female germline stem cells, and regulation of apoptosis. Eff is also a major component of Drosophila chromatin and it is particularly enriched in chromatin with repressive properties. In addition, Eff is required for telomere protection and to prevent telomere fusion. Consistent with its multiple roles in chromatin maintenance, Eff is also one of the rare factors that modulate both telomere-induced and heterochromatin-induced position effect variegation. PMID:24088712

  14. Beyond Transcription Factors: The Role of Chromatin Modifying Enzymes in Regulating Transcription Required for Memory

    ERIC Educational Resources Information Center

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately…

  15. XNP/dATRX interacts with DREF in the chromatin to regulate gene expression

    PubMed Central

    Valadez-Graham, Viviana; Yoshioka, Yasuhide; Velazquez, Oscar; Kawamori, Akihito; Vázquez, Martha; Neumann, Adina; Yamaguchi, Masamitsu; Zurita, Mario

    2012-01-01

    The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, we propose that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. We report a novel functional interaction between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression. PMID:22021382

  16. XNP/dATRX interacts with DREF in the chromatin to regulate gene expression.

    PubMed

    Valadez-Graham, Viviana; Yoshioka, Yasuhide; Velazquez, Oscar; Kawamori, Akihito; Vázquez, Martha; Neumann, Adina; Yamaguchi, Masamitsu; Zurita, Mario

    2012-02-01

    The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, we propose that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. We report a novel functional interaction between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression. PMID:22021382

  17. 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.

  18. Condensation model for the ESBWR passive condensers

    SciTech Connect

    Revankar, S. T.; Zhou, W.; Wolf, B.; Oh, S.

    2012-07-01

    In the General Electric's Economic simplified boiling water reactor (GE-ESBWR) the passive containment cooling system (PCCS) plays a major role in containment pressure control in case of an loss of coolant accident. The PCCS condenser must be able to remove sufficient energy from the reactor containment to prevent containment from exceeding its design pressure following a design basis accident. There are three PCCS condensation modes depending on the containment pressurization due to coolant discharge; complete condensation, cyclic venting and flow through mode. The present work reviews the models and presents model predictive capability along with comparison with existing data from separate effects test. The condensation models in thermal hydraulics code RELAP5 are also assessed to examine its application to various flow modes of condensation. The default model in the code predicts complete condensation well, and basically is Nusselt solution. The UCB model predicts through flow well. None of condensation model in RELAP5 predict complete condensation, cyclic venting, and through flow condensation consistently. New condensation correlations are given that accurately predict all three modes of PCCS condensation. (authors)

  19. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

    SciTech Connect

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B. . E-mail: mquintan@mail.cinvestav.mx

    2005-01-15

    Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A{sub 3} (CMA{sub 3}). Increases in DFI (15%), DFI% (4.5-fold), and CMA{sub 3} (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA{sub 3} provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

  20. Chromatin defects in normal and malformed human ejaculated and epididymal spermatozoa: a cytochemical ultrastructural study.

    PubMed

    Francavilla, S; Cordeschi, G; Gabriele, A; Gianaroli, L; Properzi, G

    1996-03-01

    Cytochemical defects in chromatin were examined by transmission electron microscopy (TEM) after the staining by alcoholic phosphotungstic acid (PTA) of normal and malformed ejaculated spermatozoa from 35 male partners of infertile couples, and in six sperm samples retrieved from the caput epididymidis of men affected by obstructive azoospermia. PTA staining was also analysed in normal ejaculates of fertile men after incubation of the washed spermatozoa with dithiothreitol (DTT) to reduce disulfides to thiols, or with DTT followed by iodoacetamide, a blocking agent for thiol groups. PTA stained 63 (27-100)% of malformed heads and 25 (10-100)% of normal sperm heads (median (range) n = 35; P = 0.0001, Wilcoxon matched pairs test). The percentage of normal heads stained by PTA was negatively correlated with the percentage of heads of normal form, with condensed chromatin and a normal acrosome (Spearman r = 0.75; P = 0.0001), and positively correlated with the percentage of malformed heads after conventional TEM analysis (Spearman r 0.60; P = 0.0001). Staining with PTA in normal heads was not correlated with the presence of non-condensed chromatin in otherwise normal sperm heads evaluated by conventional TEM analysis. In spermatozoa recovered from the caput epididymidis, 15% of normal heads were stained with PTA, significantly fewer than in ejaculated sperm samples (P = 0.014). The reduction of disulfides to thiols was associated with PTA staining of all normal heads, and this was prevented by incubation with iodoacetamide. We conclude that PTA staining of the nuclei of human ejaculated spermatozoa may indicate a defect of chromatin condensation, owing to an excess of free thiol groups. The lower percentage of normal epididymal sperm heads that stained with PTA in cases of obstructive azoospermia compared with ejaculated sperm may be related to an overoxidation of thils owing to the ageing of spermatozoa. PMID:8699409

  1. 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.

  2. Pulling chromatin apart: Unstacking or Unwrapping?

    PubMed Central

    2012-01-01

    Background Understanding the mechanical properties of chromatin is an essential step towards deciphering the physical rules of gene regulation. In the past ten years, many single molecule experiments have been carried out, and high resolution measurements of the chromatin fiber stiffness are now available. Simulations have been used in order to link those measurements with structural cues, but so far no clear agreement among different groups has been reached. Results We revisit here some of the most precise experimental results obtained with carefully reconstituted fibers. Conclusions We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by the stiffness of the DNA molecule and the 3D structure of the chromatin fiber. PMID:23186373

  3. Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

    PubMed Central

    Woodman, Julie; Hoffman, Matthew; Dzieciatkowska, Monika; Hansen, Kirk C.; Megee, Paul C.

    2015-01-01

    The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover. PMID:26354421

  4. Probabilistic modelling of chromatin code landscape reveals functional diversity of enhancer-like chromatin states

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

    Interpreting the functional state of chromatin from the combinatorial binding patterns of chromatin factors, that is, the chromatin codes, is crucial for decoding the epigenetic state of the cell. Here we present a systematic map of Drosophila chromatin states derived from data-driven probabilistic modelling of dependencies between chromatin factors. Our model not only recapitulates enhancer-like chromatin states as indicated by widely used enhancer marks but also divides these states into three functionally distinct groups, of which only one specific group possesses active enhancer activity. Moreover, we discover a strong association between one specific enhancer state and RNA Polymerase II pausing, linking transcription regulatory potential and chromatin organization. We also observe that with the exception of long-intron genes, chromatin state transition positions in transcriptionally active genes align with an absolute distance to their corresponding transcription start site, regardless of gene length. Using our method, we provide a resource that helps elucidate the functional and spatial organization of the chromatin code landscape. PMID:26841971

  5. Chromatin Dynamics During DNA Replication and Uncharacterized Replication Factors determined by Nascent Chromatin Capture (NCC) Proteomics

    PubMed Central

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Bau; Kustatscher, Georg; Nakamura, Kyosuke; de Lima Alves, Flavia; Menard, Patrice; Mejlvang, Jakob; Rappsilber, Juri; Groth, Anja

    2014-01-01

    SUMMARY To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use Nascent Chromatin Capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity-purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3995 proteins. The replication machinery and 485 chromatin factors like CAF-1, DNMT1, SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, while H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance. PMID:24561620

  6. 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

  7. Chromatin remodeling by nucleosome disassembly in vitro.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2006-02-28

    The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo. PMID:16492771

  8. 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. PMID:26208520

  9. The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo

    PubMed Central

    Ocampo, Josefina; Chereji, Răzvan V.; Eriksson, Peter R.; Clark, David J.

    2016-01-01

    Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. PMID:26861626

  10. The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo.

    PubMed

    Ocampo, Josefina; Chereji, Răzvan V; Eriksson, Peter R; Clark, David J

    2016-06-01

    Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. PMID:26861626

  11. 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

  12. Epigenomic regulation of oncogenesis by chromatin remodeling.

    PubMed

    Kumar, R; Li, D-Q; Müller, S; Knapp, S

    2016-08-25

    Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy. PMID:26804164

  13. Radiation Induced Chromatin Conformation Changes Analysed by Fluorescent Localization Microscopy, Statistical Physics, and Graph Theory

    PubMed Central

    Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W.

    2015-01-01

    It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are

  14. Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

    PubMed

    Eagen, Kyle P; Hartl, Tom A; Kornberg, Roger D

    2015-11-01

    Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. PMID:26544940

  15. Optimizing process vacuum condensers

    SciTech Connect

    Lines, J.R.; Tice, D.W.

    1997-09-01

    Vacuum condensers play a critical role in supporting vacuum processing operations. Although they may appear similar to atmospheric units, vacuum condensers have their own special designs, considerations and installation needs. By adding vacuum condensers, precondensers and intercondensers, system cost efficiency can be optimized. Vacuum-condensing systems permit reclamation of high-value product by use of a precondenser, or reduce operating costs with intercondensers. A precondenser placed between the vacuum vessel and ejector system will recover valuable process vapors and reduce vapor load to an ejector system--minimizing the system`s capital and operating costs. Similarly, an intercondenser positioned between ejector stages can condense motive steam and process vapors and reduce vapor load to downstream ejectors as well as lower capital and operating costs. The paper describes vacuum condenser systems, types of vacuum condensers, shellside condensing, tubeside condensing, noncondensable gases, precondenser pressure drop, system interdependency, equipment installation, and equipment layout.

  16. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  17. Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

    PubMed Central

    Fournier, David; Redl, Stefan; Best, Gerrit; Borsos, Máté; Tiwari, Vijay K.; Tachibana-Konwalski, Kikuë; Ketting, René F.; Parekh, Sapun H.; Cremer, Christoph; Birk, Udo J.

    2015-01-01

    During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated. PMID:26561583

  18. Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events

    PubMed Central

    Johnson, Graham D.; Jodar, Meritxell; Pique-Regi, Roger; Krawetz, Stephen A.

    2016-01-01

    Nuclear remodeling to a condensed state is a hallmark of spermatogenesis. This is achieved by replacement of histones with protamines. Regions retaining nucleosomes may be of functional significance. To determine their potential roles, sperm from wild type and transgenic mice harboring a single copy insert of the human protamine cluster were subjected to Micrococcal Nuclease-seq. CENTIPEDE, a hierarchical Bayesian model, was used to identify multiple spatial patterns, "footprints", of MNase-seq reads along the sperm genome. Regions predicted by CENTIPEDE analysis to be bound by a regulatory factor in sperm were correlated with genomic landmarks and higher order chromatin structure datasets to identify potential roles for these factors in regulating either prior or post spermatogenic, i.e., early embryonic events. This approach linked robust endogenous protamine transcription and transgene suppression to its chromatin environment within topologically associated domains. Of the candidate enhancer-bound regulatory proteins, Ctcf, was associated with chromatin domain boundaries in testes and embryonic stem cells. The continuity of Ctcf binding through the murine germline may permit rapid reconstitution of chromatin organization following fertilization. This likely reflects its preparation for early zygotic genome activation and comparatively accelerated preimplantation embryonic development program observed in mouse as compared to human and bull. PMID:27184706

  19. Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly-ADP-ribosylation

    PubMed Central

    Thomas, Colin J.; Kotova, Elena; Andrake, Mark; Adolf-Bryfogle, Jared; Glaser, Robert; Regnard, Catherine; Tulin, Alexei V.

    2014-01-01

    SUMMARY Dynamically controlled post-translational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating Poly(ADP-ribose) Polymerase 1 (PARP-1). JIL-1 phosphorylates the C-terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1 dependent genes. PMID:24508391

  20. Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle

    PubMed Central

    VANAGAS, LAURA; DALMASSO, MARIA C.; DUBREMETZ, JEAN F.; PORTIANSKY, ENRIQUE L.; OLINS, DONALD E.; ANGEL, SERGIO O.

    2014-01-01

    SUMMARY Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed. PMID:23701822

  1. Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation.

    PubMed

    Morey, Céline; Da Silva, Nelly R; Perry, Paul; Bickmore, Wendy A

    2007-03-01

    The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, we suggest that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. We show that, in the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. We conclude that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, we suggest that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts. PMID:17251268

  2. Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes.

    PubMed

    Prakash, Kirti; Fournier, David; Redl, Stefan; Best, Gerrit; Borsos, Máté; Tiwari, Vijay K; Tachibana-Konwalski, Kikuë; Ketting, René F; Parekh, Sapun H; Cremer, Christoph; Birk, Udo J

    2015-11-24

    During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated. PMID:26561583

  3. Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events.

    PubMed

    Johnson, Graham D; Jodar, Meritxell; Pique-Regi, Roger; Krawetz, Stephen A

    2016-01-01

    Nuclear remodeling to a condensed state is a hallmark of spermatogenesis. This is achieved by replacement of histones with protamines. Regions retaining nucleosomes may be of functional significance. To determine their potential roles, sperm from wild type and transgenic mice harboring a single copy insert of the human protamine cluster were subjected to Micrococcal Nuclease-seq. CENTIPEDE, a hierarchical Bayesian model, was used to identify multiple spatial patterns, "footprints", of MNase-seq reads along the sperm genome. Regions predicted by CENTIPEDE analysis to be bound by a regulatory factor in sperm were correlated with genomic landmarks and higher order chromatin structure datasets to identify potential roles for these factors in regulating either prior or post spermatogenic, i.e., early embryonic events. This approach linked robust endogenous protamine transcription and transgene suppression to its chromatin environment within topologically associated domains. Of the candidate enhancer-bound regulatory proteins, Ctcf, was associated with chromatin domain boundaries in testes and embryonic stem cells. The continuity of Ctcf binding through the murine germline may permit rapid reconstitution of chromatin organization following fertilization. This likely reflects its preparation for early zygotic genome activation and comparatively accelerated preimplantation embryonic development program observed in mouse as compared to human and bull. PMID:27184706

  4. Stacked thin layers of metaphase chromatin explain the geometry of chromosome rearrangements and banding

    PubMed Central

    Daban, Joan-Ramon

    2015-01-01

    The three-dimensional organization of tightly condensed chromatin within metaphase chromosomes has been one of the most challenging problems in structural biology since the discovery of the nucleosome. This study shows that chromosome images obtained from typical banded karyotypes and from different multicolour cytogenetic analyses can be used to gain information about the internal structure of chromosomes. Chromatin bands and the connection surfaces in sister chromatid exchanges and in cancer translocations are planar and orthogonal to the chromosome axis. Chromosome stretching produces band splitting and even the thinnest bands are orthogonal and well defined, indicating that short stretches of DNA can occupy completely the chromosome cross-section. These observations impose strong physical constraints on models that attempt to explain chromatin folding in chromosomes. The thin-plate model, which consists of many stacked layers of planar chromatin perpendicular to the chromosome axis, is compatible with the observed orientation of bands, with the existence of thin bands, and with band splitting; it is also compatible with the orthogonal orientation and planar geometry of the connection surfaces in chromosome rearrangements. The results obtained provide a consistent interpretation of the chromosome structural properties that are used in clinical cytogenetics for the diagnosis of hereditary diseases and cancers. PMID:26446309

  5. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  6. 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

  7. 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…

  8. Combined Effects of High-Dose Bisphenol A and Oxidizing Agent (KBrO3) on Cellular Microenvironment, Gene Expression, and Chromatin Structure of Ku70-deficient Mouse Embryonic Fibroblasts

    PubMed Central

    Gassman, Natalie R.; Coskun, Erdem; Jaruga, Pawel; Dizdaroglu, Miral; Wilson, Samuel H.

    2016-01-01

    Background: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBrO3). Objectives: We determined the cellular microenvironment changes induced by co-exposure of BPA and KBrO3 versus either agent alone. Methods: Ku70-deficient cells were exposed to 150 μM BPA, 20 mM KBrO3, or co-exposed to both agents. Four and 24 hr post-damage initiation by KBrO3, with BPA-only samples timed to coincide with these designated time points, we performed whole-genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH. Results: We found that 4 hr post-damage initiation, BPA exposure and co-exposure transiently condensed chromatin compared with untreated and KBrO3-only treated cells; the transcription of DNA repair proteins was also reduced. At this time point, BPA exposure and co-exposure also reduced the change in intracellular pH observed after treatment with KBrO3 alone. Twenty-four hours post-damage initiation, BPA-exposed cells showed less condensed chromatin than cells treated with KBrO3 alone; the intracellular pH of the co-exposed cells was significantly reduced compared with untreated and KBrO3-treated cells; and significant up-regulation of DNA repair proteins was observed after co-exposure. Conclusion: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku70-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the

  9. The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

    PubMed

    Altmeyer, Matthias; Toledo, Luis; Gudjonsson, Thorkell; Grøfte, Merete; Rask, Maj-Britt; Lukas, Claudia; Akimov, Vyacheslav; Blagoev, Blagoy; Bartek, Jiri; Lukas, Jiri

    2013-10-24

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling. PMID:24055346

  10. Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

    PubMed

    Waszak, Sebastian M; Delaneau, Olivier; Gschwind, Andreas R; Kilpinen, Helena; Raghav, Sunil K; Witwicki, Robert M; Orioli, Andrea; Wiederkehr, Michael; Panousis, Nikolaos I; Yurovsky, Alisa; Romano-Palumbo, Luciana; Planchon, Alexandra; Bielser, Deborah; Padioleau, Ismael; Udin, Gilles; Thurnheer, Sarah; Hacker, David; Hernandez, Nouria; Reymond, Alexandre; Deplancke, Bart; Dermitzakis, Emmanouil T

    2015-08-27

    Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain. PMID:26300124

  11. Binding of the Ah receptor to receptor binding factors in chromatin.

    PubMed

    Dunn, R T; Ruh, T S; Ruh, M F

    1993-03-01

    Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We report the binding of the Ah receptor, partially purified from rabbit liver, to receptor binding factors in chromatin. Rabbit liver chromatin proteins (CP) were isolated by adsorption of chromatin to hydroxylapatite followed by sequential extraction with 1-8 M GdnHCl. To assay for receptor binding a portion of each CP fraction was reconstituted to rabbit double-stranded DNA using a reverse gradient dialysis of 7.5 to 0 M GdnHCl. These reconstituted nucleoacidic proteins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter assay. Prior to the binding assay, [3H]TCDD-receptor complexes were partially purified by step elution from DEAE-cellulose columns. CP fractions 2, 5, and 7 were found to bind to the Ah receptor with high affinity. Scatchard analysis yielded Kd values in the nanomolar range. Competition with 2-fold excess unlabeled TCDD-receptor complexes was demonstrated, and binding was reduced markedly when the receptor was prepared in the presence of 10 mM molybdate. Such chromatin receptor binding factors (RBFs) may participate in the interaction of receptor with specific DNA sequences resulting in modulation of specific gene expression. PMID:8384852

  12. The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

    PubMed Central

    Lake, Robert J.; Boetefuer, Erica L.; Won, Kyoung-Jae; Fan, Hua-Ying

    2016-01-01

    Cockayne syndrome is a premature aging disease associated with numerous developmental and neurological abnormalities, and elevated levels of reactive oxygen species have been found in cells derived from Cockayne syndrome patients. The majority of Cockayne syndrome cases contain mutations in the ATP-dependent chromatin remodeler CSB; however, how CSB protects cells from oxidative stress remains largely unclear. Here, we demonstrate that oxidative stress alters the genomic occupancy of the CSB protein and increases CSB occupancy at promoters. Additionally, we found that the long-range chromatin-structure regulator CTCF plays a pivotal role in regulating sites of genomic CSB occupancy upon oxidative stress. We show that CSB directly interacts with CTCF in vitro and that oxidative stress enhances the CSB-CTCF interaction in cells. Reciprocally, we demonstrate that CSB facilitates CTCF-DNA interactions in vitro and regulates CTCF-chromatin interactions in oxidatively stressed cells. Together, our results indicate that CSB and CTCF can regulate each other's chromatin association, thereby modulating chromatin structure and coordinating gene expression in response to oxidative stress. PMID:26578602

  13. Condensate Mixtures and Tunneling

    SciTech Connect

    Timmermans, E.

    1998-09-14

    The experimental study of condensate mixtures is a particularly exciting application of the recently developed atomic-trap Bose-Einstein condensate (BEC) technology: such multiple condensates represent the first laboratory systems of distinguishable boson superfluid mixtures. In addition, as the authors point out in this paper, the possibility of inter-condensate tunneling greatly enhances the richness of the condensate mixture physics. Not only does tunneling give rise to the oscillating particle currents between condensates of different chemical potentials, such as those studied extensively in the condensed matter Josephson junction experiments, it also affects the near-equilibrium dynamics and stability of the condensate mixtures. In particular, the stabilizing influence of tunneling with respect to spatial separation (phase separation) could be of considerable practical importance to the atomic trap systems. Furthermore, the creation of mixtures of atomic and molecular condensates could introduce a novel type of tunneling process, involving the conversion of a pair of atomic condensate bosons into a single molecular condensate boson. The static description of condensate mixtures with such type of pair tunneling suggests the possibility of observing dilute condensates with the liquid-like property of a self-determined density.

  14. MARCC (Matrix-Assisted Reader Chromatin Capture): an antibody-free method to enrich and analyze combinatorial nucleosome modifications

    PubMed Central

    Su, Zhangli

    2016-01-01

    Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody-free method named MARCC (Matrix-Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin-interacting protein modules, or so-called reader domains, which can bind in a combinatorial modification-dependent manner. The enriched chromatin can be quantified by western blotting or mass spectrometry for the co-existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next-generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications. PMID:26131849

  15. Targeting chromatin to improve radiation response

    PubMed Central

    Olcina, M M; O'Dell, S

    2015-01-01

    Chromatin, the structure formed by the wrapping of approximately 146 base pairs of DNA around an octamer of histones, has a profound impact on numerous DNA-based processes. Chromatin modifications and chromatin remodellers have recently been implicated in important aspects of the DNA damage response including facilitating the initial sensing of the damage as well as subsequent recruitment of repair factors. Radiation is an effective cancer therapy for a large number of tumours, and there is considerable interest in finding approaches that might further increase the efficacy of radiotherapy. The use of radiation leads to the generation of DNA damage and, therefore, agents that can affect the sensing and repair of DNA damage may have an impact on overall radiation efficacy. The chromatin modifications as well as chromatin modifiers that have been associated with the DNA damage response will be summarized in this review. An emphasis will be placed on those processes that can be pharmacologically manipulated with currently available inhibitors. The rationale for the use of these inhibitors in combination with radiation will also be described. PMID:25513745

  16. PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues

    PubMed Central

    Yamaguchi, Nobutoshi; Winter, Cara M.; Wu, Miin-Feng; Kwon, Chang Seob; William, Dilusha A.; Wagner, Doris

    2014-01-01

    The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days. PMID:24653666

  17. 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

  18. Chromatin remodeling in cardiovascular development and physiology

    PubMed Central

    Han, Pei; Hang, Calvin T.; Yang, Jin; Chang, Ching-Pin

    2010-01-01

    Chromatin regulation provides an important means of controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various transcription factors in the fine regulation of gene expression, essential for all aspects of cardiovascular biology. Aberrant cardiac gene expression, triggered by a variety of pathological insults, can cause heart diseases in both animals and humans. The severity of cardiomyopathy and heart failure correlates strongly with abnormal cardiac gene expression. Therefore, controlling cardiac gene expression presents a promising approach to the treatment of human cardiomyopathy. This review focuses on the roles of ATP-dependent chromatin-remodeling factors and chromatin-modifying enzymes in the control of gene expression during cardiovascular development and disease. PMID:21293009

  19. Condensates in Jovian Atmospheres

    NASA Technical Reports Server (NTRS)

    West, R.

    1999-01-01

    Thermochemical equilibrium theory which starts with temperature/pressure profiles, compositional information and thermodynamic data for condensable species in the jovian planet atmospheres predicts layers of condensate clouds in the upper troposphere.

  20. Structure of RCC1 chromatin factor bound to the nucleosome core particle

    SciTech Connect

    Makde, Ravindra D.; England, Joseph R.; Yennawar, Hemant P.; Tan, Song

    2010-11-11

    The small GTPase Ran enzyme regulates critical eukaryotic cellular functions including nuclear transport and mitosis through the creation of a RanGTP gradient around the chromosomes. This concentration gradient is created by the chromatin-bound RCC1 (regulator of chromosome condensation) protein, which recruits Ran to nucleosomes and activates Ran's nucleotide exchange activity. Although RCC1 has been shown to bind directly with the nucleosome, the molecular details of this interaction were not known. Here we determine the crystal structure of a complex of Drosophila RCC1 and the nucleosome core particle at 2.9 {angstrom} resolution, providing an atomic view of how a chromatin protein interacts with the histone and DNA components of the nucleosome. Our structure also suggests that the Widom 601 DNA positioning sequence present in the nucleosomes forms a 145-base-pair nucleosome core particle, not the expected canonical 147-base-pair particle.

  1. Nucleosome dynamics during chromatin remodeling in vivo

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    ABSTRACT 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. PMID:26933790

  2. Bacterial chromatin: converging views at different scales.

    PubMed

    Dame, Remus T; Tark-Dame, Mariliis

    2016-06-01

    Bacterial genomes are functionally organized and compactly folded into a structure referred to as bacterial chromatin or the nucleoid. An important role in genome folding is attributed to Nucleoid-Associated Proteins, also referred to as bacterial chromatin proteins. Although a lot of molecular insight in the mechanisms of operation of these proteins has been generated in the test tube, knowledge on genome organization in the cellular context is still lagging behind severely. Here, we discuss important advances in the understanding of three-dimensional genome organization due to the application of Chromosome Conformation Capture and super-resolution microscopy techniques. We focus on bacterial chromatin proteins whose proposed role in genome organization is supported by these approaches. Moreover, we discuss recent insights into the interrelationship between genome organization and genome activity/stability in bacteria. PMID:26942688

  3. 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

  4. 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

  5. Open chromatin reveals the functional maize genome.

    PubMed

    Rodgers-Melnick, Eli; Vera, Daniel L; Bass, Hank W; Buckler, Edward S

    2016-05-31

    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

  6. 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

  7. Nuclease digestion studies of chromatin structure

    SciTech Connect

    Deutsch, S.M.

    1987-01-01

    Micrococcal nuclease, which preferentially cleaves linker DNA in chromatin, was immobilized by covalent attachment to CNBr-activated agarose beads and used to study the accessibility of linker DNA in chromatin fibers prepared from chicken erythrocyte nuclei. This immobilized nuclease was able to cleave chromatin fibers into the typical pattern of fragments corresponding to multiples of mononucleosomes. Cleavage from only the ends of the fibers was ruled out by examining the products of cleavage of fibers end-labelled with /sup 35/P. Comparison of the rate of digestion by immobilized and soluble micrococcal nuclease indicated that the fiber structure does not significantly affect access to linker DNA. The absence of an effect of reducing temperatures on the rate of digestion of fibers, as compared to short oligonucleosomes, indicated that breathing motions to allow access to the fiber interior were not required for cleavage of linker DNA.

  8. [Comparative characteristics of chromatin endonuclease fragments].

    PubMed

    Miul'berg, A A; Tishchenko, L I; Domkina, L K

    1977-05-01

    Soluble fragments of chromatin obtained by Ca, Mg-dependent endonuclease digest of rat liver nuclei, have been separated by gel chromatography on Sepharose 4B into three zones, containing oligomers, tetramers--dimers and monomers, respectively. The content of nonhistone proteins and particularly lysine-rich histones is decreased with a transition from theoligomers to monomers. The average protein/DNA ratio of the monomers is equal to 1.36 and that of histone/DNA ratio--to 0.82. The dependence of the degree of chromatin digest by endonuclease on its protein content and conditions of isolation and incubation of nuclei is discussed. The chromatin monomer formed appears to be made up of a nucleosome and short portions of spacer DNA bound to some part of histone HI and nonhistone proteins. PMID:889964

  9. 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

  10. Linker Histones Incorporation Maintains Chromatin Fiber Plasticity

    PubMed Central

    Recouvreux, Pierre; Lavelle, Christophe; Barbi, Maria; Conde e Silva, Natalia; Le Cam, Eric; Victor, Jean-Marc; Viovy, Jean-Louis

    2011-01-01

    Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones. PMID:21641318

  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. PMID:26933790

  12. Chromatin organization during spermiogenesis in Octopus vulgaris. II: DNA-interacting proteins.

    PubMed

    Giménez-Bonafé, Pepita; Soler, Fina Martínez; Buesa, Carlos; Sautière, Pierre-Eric; Ausió, Juan; Kouach, Mostafa; Kasinsky, Harold E; Chiva, Manel

    2004-06-01

    In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris. PMID:15095345

  13. Dynamic reprogramming of chromatin: paradigmatic palimpsests and HES factors

    PubMed Central

    Kok, Kurtulus; Arnosti, David N.

    2015-01-01

    Temporal and spatial control of transcription in development is dictated to a great extent by transcriptional repressors. Some repressor complexes, such as Polycomp-group proteins, induce relatively long-term non-permissive states, whereas others such as hairy/enhancer of split (HES) family repressors are linked to dynamically modulated chromatin states associated with cycling expression of target genes. The mode of action and specificity of repressors involved in mediating this latter form of epigenetic control are unknown. Oscillating expression of HES repressors controlled by signaling pathways such as Notch suggests that the entire ensemble of HES–associated co-repressors and histone modifying complexes readily cycle on and off genes. Dynamic interactions between these factors and chromatin seem to be crucial in maintaining multipotency of progenitor cells, but the significance of such interactions in more differentiated cells is less well understood. We discuss here how genome-wide analyses and real-time gene expression measurements of HES regulated genes can help decipher the detailed mechanisms and biological importance of highly dynamic transcriptional switching mediated by epigenetic changes. PMID:25713582

  14. Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder

    SciTech Connect

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat; Chakraborty, Payal; Jana, Kuladip; Dasgupta, Dipak

    2015-07-10

    Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a ‘dual binder’. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications. - Highlights: • Ellipticine acts a dual binder binding to both DNA and core histone(s). • It induces structural perturbations in chromatin, chromatosome and histone octamer. • It alters histones acetylation and affects global gene expression.

  15. Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin.

    PubMed

    Zhou, Zhongwu; Yang, Yi; Konieczny, Stephen F; Irudayaraj, Joseph M K

    2015-12-24

    An ultra fast and unbiased method that uses salicylic acid coated magnetic nanoparticles (SAMNPs) and magnetophoretic chromatography is developed to extract chromatin associated RNAs (CARs). The SAMNPs were first used for enriching cells from the cell culture media and further used for capturing chromatin after cells were lysed. The formed SAMNPs-chromatin complexes were transferred to a viscous polyethylene glycol (PEG) solution stored in a 200-μl pipette tip. Due to the difference in viscosities, a bi-layer liquid was formed inside the pipette tip. The SAMNPs-chromatin complexes were separated from the free SAMNPs and free RNA-SAMNPs complexes by applying an external magnetic field. The CARs were further extracted from the SAMNP-chromatin complexes directly. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h. PMID:26643718

  16. Chromatin higher-order structures and gene regulation

    PubMed Central

    Li, Guohong

    2011-01-01

    Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a critical role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation. PMID:21342762

  17. 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.

  18. Chromosomes without a 30-nm chromatin fiber

    PubMed Central

    Joti, Yasumasa; Hikima, Takaaki; Nishino, Yoshinori; Kamada, Fukumi; Hihara, Saera; Takata, Hideaki; Ishikawa, Tetsuya; Maeshima, Kazuhiro

    2012-01-01

    How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure. PMID:22825571

  19. Chromatin-modifying and -remodeling complexes.

    PubMed

    Kornberg, R D; Lorch, Y

    1999-04-01

    Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure. PMID:10322131

  20. Monte Carlo simulation of chromatin stretching.

    PubMed

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

    2006-04-01

    We present Monte Carlo (MC) simulations of the stretching of a single 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 degrees to 130 degrees increases the stiffness significantly. An increase in the opening angle from 22 degrees to 34 degrees 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. PMID:16711856

  1. 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.

  2. EBV latency types adopt alternative chromatin conformations.

    PubMed

    Tempera, Italo; Klichinsky, Michael; Lieberman, Paul M

    2011-07-01

    Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer. PMID:21829357

  3. Chromatin organization of gammaherpesvirus latent genomes.

    PubMed

    Tempera, Italo; Lieberman, Paul M

    2010-01-01

    The gammaherpesviruses are a subclass of the herpesvirus family that establish stable latent infections in proliferating lymphoid and epithelial cells. The latent genomes are maintained as multicopy chromatinized episomes that replicate in synchrony with the cellular genome. Importantly, most of the episomes do not integrate into the host chromosome. Therefore, it is essential that the viral "minichromosome" establish a chromatin structure that is suitable for gene expression, DNA replication, and chromosome segregation. Evidence suggests that chromatin organization is important for each of these functions and plays a regulatory role in the establishment and maintenance of latent infection. Here, we review recent studies on the chromatin organization of the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). We discuss the potential role of viral origins of DNA replication and viral encoded origin-binding proteins like EBNA1 and LANA in establishment of viral chromosome organization during latent infection. We also discuss the roles of host cell factors, like CTCF and cohesins, that contribute to higher-order chromosome structures that may be important for stable gene expression programs during latent infection in proliferating cells. PMID:19853673

  4. 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.

  5. Anterior-posterior differences in HoxD chromatin topology in limb development

    PubMed Central

    Williamson, Iain; Eskeland, Ragnhild; Lettice, Laura A.; Hill, Alison E.; Boyle, Shelagh; Grimes, Graeme R.; Hill, Robert E.; Bickmore, Wendy A.

    2012-01-01

    A late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5′ Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5′ HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5′ HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb). PMID:22872084

  6. Inferential modeling of 3D chromatin structure

    PubMed Central

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-01-01

    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. PMID:25690896

  7. Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation

    PubMed Central

    Alvarez-Saavedra, Matías; De Repentigny, Yves; Lagali, Pamela S.; Raghu Ram, Edupuganti V. S.; Yan, Keqin; Hashem, Emile; Ivanochko, Danton; Huh, Michael S.; Yang, Doo; Mears, Alan J.; Todd, Matthew A. M.; Corcoran, Chelsea P.; Bassett, Erin A.; Tokarew, Nicholas J. A.; Kokavec, Juraj; Majumder, Romit; Ioshikhes, Ilya; Wallace, Valerie A.; Kothary, Rashmi; Meshorer, Eran; Stopka, Tomas; Skoultchi, Arthur I.; Picketts, David J.

    2014-01-01

    Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation. PMID:24946904

  8. Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation

    PubMed Central

    Jossé, Rozenn; Zhang, Yong-Wei; Giroux, Valentin; Ghosh, Arun K.; Luo, Ji; Pommier, Yves

    2015-01-01

    Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt’s lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1−/− MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA. PMID:26058013

  9. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R. . E-mail: tsaneva@biochem.ucl.ac.uk

    2005-11-01

    TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

  10. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

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

    2016-07-01

    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. PMID:27391925

  11. Chromatin signature discovery via histone modification profile alignments

    PubMed Central

    Wang, Jianrong; Lunyak, Victoria V.; Jordan, I. King

    2012-01-01

    We report on the development of an unsupervised algorithm for the genome-wide discovery and analysis of chromatin signatures. Our Chromatin-profile Alignment followed by Tree-clustering algorithm (ChAT) employs dynamic programming of combinatorial histone modification profiles to identify locally similar chromatin sub-regions and provides complementary utility with respect to existing methods. We applied ChAT to genomic maps of 39 histone modifications in human CD4+ T cells to identify both known and novel chromatin signatures. ChAT was able to detect chromatin signatures previously associated with transcription start sites and enhancers as well as novel signatures associated with a variety of regulatory elements. Promoter-associated signatures discovered with ChAT indicate that complex chromatin signatures, made up of numerous co-located histone modifications, facilitate cell-type specific gene expression. The discovery of novel L1 retrotransposon-associated bivalent chromatin signatures suggests that these elements influence the mono-allelic expression of human genes by shaping the chromatin environment of imprinted genomic regions. Analysis of long gene-associated chromatin signatures point to a role for the H4K20me1 and H3K79me3 histone modifications in transcriptional pause release. The novel chromatin signatures and functional associations uncovered by ChAT underscore the ability of the algorithm to yield novel insight on chromatin-based regulatory mechanisms. PMID:22989711

  12. miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy

    PubMed Central

    Badal, Shawn S.; Wang, Yin; Long, Jianyin; Corcoran, David L.; Chang, Benny H.; Truong, Luan D.; Kanwar, Yashpal S.; Overbeek, Paul A.; Danesh, Farhad R.

    2016-01-01

    How the kidney responds to the metabolic cues from the environment remains a central question in kidney research. This question is particularly relevant to the pathogenesis of diabetic nephropathy (DN) in which evidence suggests that metabolic events in podocytes regulate chromatin structure. Here, we show that miR-93 is a critical metabolic/epigenetic switch in the diabetic milieu linking the metabolic state to chromatin remodelling. Mice with inducible overexpression of a miR-93 transgene exclusively in podocytes exhibit significant improvements in key features of DN. We identify miR-93 as a regulator of nucleosomal dynamics in podocytes. miR-93 has a critical role in chromatin reorganization and progression of DN by modulating its target Msk2, a histone kinase, and its substrate H3S10. These findings implicate a central role for miR-93 in high glucose-induced chromatin remodelling in the kidney, and provide evidence for a previously unrecognized role for Msk2 as a target for DN therapy. PMID:27350436

  13. Barrier-to-autointegration factor (BAF) involvement in prelamin A-related chromatin organization changes.

    PubMed

    Loi, Manuela; Cenni, Vittoria; Duchi, Serena; Squarzoni, Stefano; Lopez-Otin, Carlos; Foisner, Roland; Lattanzi, Giovanna; Capanni, Cristina

    2016-03-29

    Chromatin disorganization is one of the major alterations linked to prelamin A processing impairment. In this study we demonstrate that BAF is necessary to modulate prelamin A effects on chromatin structure. We show that when prelamin A and BAF cannot properly interact no prelamin A-dependent effects on chromatin occur; similar to what is observed in human Nestor Guillermo Progeria Syndrome cells harboring a BAF mutation, in HEK293 cells expressing a BAF mutant unable to bind prelamin A, or in siRNA mediated BAF-depleted HEK293 cells expressing prelamin A. BAF is necessary to induce histone trimethyl-H3K9 as well as HP1-alpha and LAP2-alpha nuclear relocalization in response to prelamin A accumulation. These findings are enforced by electron microscopy evaluations showing how the prelamin A-BAF interaction governs overall chromatin organization. Finally, we demonstrate that the LAP2-alpha nuclear localization defect observed in HGPS cells involves the progerin-BAF interaction, thus establishing a functional link between BAF and prelamin A pathological forms. PMID:26701887

  14. The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis.

    PubMed

    Serber, Daniel W; Runge, John S; Menon, Debashish U; Magnuson, Terry

    2016-01-01

    The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination. PMID:26607718

  15. Barrier-to-Autointegration Factor (BAF) involvement in prelamin A-related chromatin organization changes

    PubMed Central

    Loi, Manuela; Cenni, Vittoria; Duchi, Serena; Squarzoni, Stefano; Lopez-Otin, Carlos; Foisner, Roland; Lattanzi, Giovanna; Capanni, Cristina

    2016-01-01

    Chromatin disorganization is one of the major alterations linked to prelamin A processing impairment. In this study we demonstrate that BAF is necessary to modulate prelamin A effects on chromatin structure. We show that when prelamin A and BAF cannot properly interact no prelamin A-dependent effects on chromatin occur; similar to what is observed in human Nestor Guillermo Progeria Syndrome cells harboring a BAF mutation, in HEK293 cells expressing a BAF mutant unable to bind prelamin A, or in siRNA mediated BAF-depleted HEK293 cells expressing prelamin A. BAF is necessary to induce histone trimethyl-H3K9 as well as HP1-alpha and LAP2-alpha nuclear relocalization in response to prelamin A accumulation. These findings are enforced by electron microscopy evaluations showing how the prelamin A-BAF interaction governs overall chromatin organization. Finally, we demonstrate that the LAP2-alpha nuclear localization defect observed in HGPS cells involves the progerin-BAF interaction, thus establishing a functional link between BAF and prelamin A pathological forms. PMID:26701887

  16. The Effect of Internucleosomal Interaction on Folding of the Chromatin Fiber

    PubMed Central

    Stehr, René; Kepper, Nick; Rippe, Karsten; Wedemann, Gero

    2008-01-01

    The folding of the nucleosome chain into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The fiber conformation depends crucially on the interaction between individual nucleosomes. However, this parameter has not been accurately determined experimentally, and it is affected by posttranslational histone modifications and binding of chromosomal proteins. Here, the effect of different internucleosomal interaction strengths on the fiber conformation was investigated by Monte Carlo computer simulations. The fiber geometry was modeled to fit that of chicken erythrocyte chromatin, which has been examined in numerous experimental studies. In the Monte Carlo simulation, the nucleosome shape was described as an oblate spherocylinder, and a replica exchange protocol was developed to reach thermal equilibrium for a broad range of internucleosomal interaction energies. The simulations revealed the large impact of the nucleosome geometry and the nucleosome repeat length on the compaction of the chromatin fiber. At high internucleosomal interaction energies, a lateral self-association of distant fiber parts and an interdigitation of nucleosomes were apparent. These results identify key factors for the control of the compaction and higher order folding of the chromatin fiber. PMID:18658212

  17. miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy.

    PubMed

    Badal, Shawn S; Wang, Yin; Long, Jianyin; Corcoran, David L; Chang, Benny H; Truong, Luan D; Kanwar, Yashpal S; Overbeek, Paul A; Danesh, Farhad R

    2016-01-01

    How the kidney responds to the metabolic cues from the environment remains a central question in kidney research. This question is particularly relevant to the pathogenesis of diabetic nephropathy (DN) in which evidence suggests that metabolic events in podocytes regulate chromatin structure. Here, we show that miR-93 is a critical metabolic/epigenetic switch in the diabetic milieu linking the metabolic state to chromatin remodelling. Mice with inducible overexpression of a miR-93 transgene exclusively in podocytes exhibit significant improvements in key features of DN. We identify miR-93 as a regulator of nucleosomal dynamics in podocytes. miR-93 has a critical role in chromatin reorganization and progression of DN by modulating its target Msk2, a histone kinase, and its substrate H3S10. These findings implicate a central role for miR-93 in high glucose-induced chromatin remodelling in the kidney, and provide evidence for a previously unrecognized role for Msk2 as a target for DN therapy. PMID:27350436

  18. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

    PubMed

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-12-17

    Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. PMID:26686651

  19. Focus on the centre: the role of chromatin on the regulation of centromere identity and function

    PubMed Central

    Torras-Llort, Mònica; Moreno-Moreno, Olga; Azorín, Fernando

    2009-01-01

    The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere-specific histone H3 variant CenH3, originally called CENP-A, which replaces canonical histone H3 at centromeres. CenH3-chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3-chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly. PMID:19629040

  20. 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.

  1. Microscopic imaging of DNA condensation in the presence of charged nanospheres

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Sandhu, Tejdev; Nordlund, Thomas

    2004-11-01

    DNA forms condensates in specific environments. In chromatin, DNA is in condensed form. DNA becomes compact by winding around positively-charged proteins called histones. Negatively-charged DNA phosphates interact electrostatically with histones and wind over them. To model the complex process of chromatin formation in cells, λ -phage (16μ m long) and herring sperm (variable length) DNAs are allowed to interact with nanospheres of size 40nm and 930nm containing 1.8x10^4 and 2.6x10^8 positive surface charges respectively at pH 7.5, to form condensates without any enzyme action. Formation of DNA condensates are imaged at various concentrations, pH's, viscosities, and ionic strengths. Images of condensate in 10-20% glycerol, which has 1.3-1.8 times the viscosity of water show smaller aggregates of size 2-4μ m in contrast to larger aggregates of size 10-50μ m formed in aqueous buffer, which indicates viscosity plays a major role in formation of these condensates. Decreases in the concentration of DNA and spheres cause decrease in the size and number of aggregates. Presence of DNA in the condensate is confirmed by the fluorescence emission from YOYO-1-iodide. We present a simple electrostatic model for this aggregation process.

  2. Calorie restriction and the exercise of chromatin

    PubMed Central

    Vaquero, Alejandro; Reinberg, Danny

    2009-01-01

    Since the earliest stages of evolution, organisms have faced the challenge of sensing and adapting to environmental changes for their survival under compromising conditions such as food depletion or stress. Implicit in these responses are mechanisms developed during evolution that include the targeting of chromatin to allow or prevent expression of fundamental genes and to protect genome integrity. Among the different approaches to study these mechanisms, the analysis of the response to a moderate reduction of energy intake, also known as calorie restriction (CR), has become one of the best sources of information regarding the factors and pathways involved in metabolic adaptation from lower to higher eukaryotes. Furthermore, responses to CR are involved in life span regulation—conserved from yeast to mammals—and therefore have garnered major research interest. Herein we review current knowledge of responses to CR at the molecular level and their functional link to chromatin. PMID:19608767

  3. Intergenic Locations of Rice Centromeric Chromatin

    PubMed Central

    Yan, Huihuang; Talbert, Paul B; Lee, Hye-Ran; Jett, Jamie; Henikoff, Steven; Chen, Feng; Jiang, Jiming

    2008-01-01

    Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions. PMID:19067486

  4. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

    He, Yan; Wang, Minghui; Sun, Qi; Pawlowski, Wojciech P

    2016-01-01

    Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi. PMID:27511175

  5. On the topology of chromatin fibres

    PubMed Central

    Barbi, Maria; Mozziconacci, Julien; Victor, Jean-Marc; Wong, Hua; Lavelle, Christophe

    2012-01-01

    The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (twist, writhe and linking number) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed. PMID:24098838

  6. Adult human arterial smooth muscle cells in primary culture. Modulation from contractile to synthetic phenotype.

    PubMed

    Thyberg, J; Nilsson, J; Palmberg, L; Sjölund, M

    1985-01-01

    Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1-2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3-4 days. It was accompanied by initiation of DNA replication and mitosis. The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis. PMID:3967287

  7. 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.

  8. 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.

  9. Measure Guideline: Evaporative Condensers

    SciTech Connect

    German, A; Dakin, B.; Hoeschele, M.

    2012-03-01

    This measure guideline on evaporative condensers provides information on properly designing, installing, and maintaining evaporative condenser systems as well as understanding the benefits, costs, and tradeoffs. This is a prescriptive approach that outlines selection criteria, design and installation procedures, and operation and maintenance best practices.

  10. 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

  11. The role of chromatin conformations in diffusional transport of chromatin-binding proteins: Cartesian lattice simulations

    NASA Astrophysics Data System (ADS)

    Wedemeier, Annika; Zhang, Ting; Merlitz, Holger; Wu, Chen-Xu; Langowski, Jörg

    2008-04-01

    In this paper, a lattice model for the diffusional transport of chromatin-binding particles in the interphase cell nucleus is proposed. Sliding effects are studied in dense networks of chromatin fibers created by three different methods: Randomly distributed, noninterconnected obstacles, a random walk chain model with an attractive step potential, and a self-avoiding random walk chain model with a hard repulsive core and attractive surroundings. By comparing a discrete and continuous version of the random walk chain model, we demonstrate that lattice discretization does not alter the diffusion of chromatin-binding particles. The influence of conformational properties of the fiber network on the particle sliding is investigated in detail while varying occupation volume, sliding probability, chain length, and persistence length. It is observed that adjacency of the monomers, the excluded volume effect incorporated in the self-avoiding random walk model, and the persistence length affect the chromatin-binding particle diffusion. It is demonstrated that sliding particles sense local chain structures. When plotting the diffusion coefficient as a function of the accessible volume for diffusing particles, the data fall onto master curves depending on the persistence length. However, once intersegment transfer is involved, chromatin-binding proteins no longer perceive local chain structures.

  12. Geothermal steam condensate reinjection

    NASA Technical Reports Server (NTRS)

    Chasteen, A. J.

    1974-01-01

    Geothermal electric generating plants which use condensing turbines and generate and excess of condensed steam which must be disposed of are discussed. At the Geysers, California, the largest geothermal development in the world, this steam condensate has been reinjected into the steam reservoir since 1968. A total of 3,150,000,000 gallons of steam condensate has been reinjected since that time with no noticeable effect on the adjacent producing wells. Currently, 3,700,000 gallons/day from 412 MW of installed capacity are being injected into 5 wells. Reinjection has also proven to be a satisfactory method of disposing of geothermal condensate a Imperial Valley, California, and at the Valles Caldera, New Mexico.

  13. Freeze-Tolerant Condensers

    NASA Technical Reports Server (NTRS)

    Crowley, Christopher J.; Elkouhk, Nabil

    2004-01-01

    Two condensers designed for use in dissipating heat carried by working fluids feature two-phase, self-adjusting configurations such that their working lengths automatically vary to suit their input power levels and/or heat-sink temperatures. A key advantage of these condensers is that they can function even if the temperatures of their heat sinks fall below the freezing temperatures of their working fluids and the fluids freeze. The condensers can even be restarted from the frozen condition. The top part of the figure depicts the layout of the first condenser. A two-phase (liquid and vapor) condenser/vapor tube is thermally connected to a heat sink typically, a radiatively or convectively cooled metal panel. A single-phase (liquid) condensate-return tube (return artery) is also thermally connected to the heat sink. At intervals along their lengths, the condenser/vapor tube and the return artery are interconnected through porous plugs. This condenser configuration affords tolerance of freezing, variable effective thermal conductance (such that the return temperature remains nearly constant, independently of the ultimate sink temperature), and overall pressure drop smaller than it would be without the porous interconnections. An additional benefit of this configuration is that the condenser can be made to recover from the completely frozen condition either without using heaters, or else with the help of heaters much smaller than would otherwise be needed. The second condenser affords the same advantages and is based on a similar principle, but it has a different configuration that affords improved flow of working fluid, simplified construction, reduced weight, and faster recovery from a frozen condition.

  14. Relationship Between Chromatin Structure and Sensitivity to Molecularly Targeted Auger Electron Radiation Therapy

    SciTech Connect

    Terry, Samantha Y.A.

    2012-07-15

    Purpose: The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated. Methods and Materials: Chromatin structure was altered in MDA-MB-468 and 231-H2N human breast cancer cells by suberoylanilide hydroxamic acid (SAHA), 5-aza-2-deoxycytidine, or hypertonic treatment. The extent and duration of chromatin structural changes were evaluated using the micrococcal nuclease assay. DNA damage ({gamma}H2AX assay) and clonogenic survival were evaluated after exposure to {sup 111}In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of {sup 111}In-DTPA-hEGF after chromatin modification was investigated in cell fractionation experiments. Results: Chromatin remained condensed for up to 20 minutes after NaCl and in a relaxed state 24 hours after SAHA treatment. The number of {gamma}H2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 {mu}M) compared with IR alone (16 {+-} 0.6 and 14 {+-} 0.3 vs. 12 {+-} 0.4 and 11 {+-} 0.2, respectively). More {gamma}H2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to {sup 111}In-DTPA-hEGF (6 MBq/{mu}g) plus SAHA vs. {sup 111}In-DTPA-hEGF alone (11 {+-} 0.3 and 12 {+-} 0.7 vs. 9 {+-} 0.4 and 7 {+-} 0.3, respectively). 5-aza-2-deoxycytidine enhanced the DNA damage caused by IR and {sup 111}In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 {mu}M) vs. IR alone (0.6% {+-} 0.01 and 0.3% {+-} 0.2 vs. 5.8% {+-} 0.2 and 2% {+-} 0.1, respectively) and after {sup 111}In-DTPA-hEGF plus SAHA compared to {sup 111}In-DTPA-hEGF alone (21% {+-} 0.4% and 19% {+-} 4.6 vs. 33% {+-} 2.3 and 32% {+-} 3.7). SAHA did not affect {sup 111}In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer {gamma}H2AX foci per cell

  15. Alpha Condensates in Atomic Nuclei

    SciTech Connect

    Suzuki, Y.; Matsumura, H.

    2005-11-21

    Recent issues on Bose-Einstein condensation (BEC) of {alpha}-particles in nuclei are reviewed. A candidate of condensates is discussed for some states in 12C and 16O by defining the amount of {alpha} condensation.

  16. Small Chemical Chromatin Effectors Alter Secondary Metabolite Production in Aspergillus clavatus

    PubMed Central

    Zutz, Christoph; Gacek, Agnieszka; Sulyok, Michael; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

    2013-01-01

    The filamentous fungus Aspergillus clavatus is known to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A, and cytochalasin E. In fungi, the production of most SM is strongly influenced by environmental factors and nutrients. Furthermore, it has been shown that the regulation of SM gene clusters is largely based on modulation of a chromatin structure. Communication between fungi and bacteria also triggers chromatin-based induction of silent SM gene clusters. Consequently, chemical chromatin effectors known to inhibit histone deacetylases (HDACs) and DNA-methyltransferases (DNMTs) influence the SM profile of several fungi. In this study, we tested the effect of five different chemicals, which are known to affect chromatin structure, on SM production in A. clavatus using two growth media with a different organic nitrogen source. We found that production of patulin was completely inhibited and cytochalasin E levels strongly reduced, whereas growing A. clavatus in media containing soya-derived peptone led to substantially higher pseurotin A levels. The HDAC inhibitors valproic acid, trichostatin A and butyrate, as well as the DNMT inhibitor 5-azacytidine (AZA) and N-acetyl-d-glucosamine, which was used as a proxy for bacterial fungal co-cultivation, had profound influence on SM accumulation and transcription of the corresponding biosynthetic genes. However, the repressing effect of the soya-based nitrogen source on patulin production could not be bypassed by any of the small chemical chromatin effectors. Interestingly, AZA influenced some SM cluster genes and SM production although no Aspergillus species has yet been shown to carry detectable DNA methylation. PMID:24105402

  17. Genetic variants in chromatin-remodeling pathway associated with lung cancer risk in a Chinese population.

    PubMed

    Geng, Liguo; Zhu, Meng; Wang, Yuzhuo; Cheng, Yang; Liu, Jia; Shen, Wei; Li, Zhihua; Zhang, Jiahui; Wang, Cheng; Jin, Guangfu; Ma, Hongxia; Shen, Hongbing; Hu, Zhibin; Dai, Juncheng

    2016-08-10

    Chromatin remodeling complexes utilize the energy of ATP hydrolysis to remodel nucleosomes and have essential roles in transcriptional modulation. Increasing evidences indicate that these complexes directly interact with numerous proteins and regulate the formation of cancer. However, few studies reported the association of polymorphisms in chromatin remodeling genes and lung cancer. We hypothesized that variants in critical genes of chromatin remodeling pathway might contribute to the susceptibility of lung cancer. To validate this hypothesis, we systematically screened 40 polymorphisms in six key chromatin remodeling genes (SMARCA5, SMARCC2, SMARCD2, ARID1A, NR3C1 and SATB1) and evaluated them with a case-control study including 1341 cases and 1982 controls. Logistic regression revealed that four variants in NR3C1 and SATB1 were significantly associated with lung cancer risk after false discovery rate (FDR) correction [For NR3C1, rs9324921: odds ratio (OR)=1.23, P for FDR=0.029; rs12521436: OR=0.85, P for FDR=0.040; rs4912913: OR=1.17, P for FDR=0.040; For SATB1, rs6808523: OR=1.33, P for FDR=0.040]. Combing analysis presented a significant allele-dosage tendency for the number of risk alleles and lung cancer risk (Ptrend<0.001). Moreover, expression quantitative trait loci (eQTL) analysis revealed that these two genes were differently expressed between lung tumor and adjacent normal tissues in the database of The Cancer Genome Atlas (TCGA) (P=0.009 for rs6808523). These findings suggested that genetic variants in key chromatin remodeling genes may contribute to lung cancer risk in Chinese population. Further large and well-designed studies are warranted to validate our results. PMID:27179949

  18. Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

    PubMed Central

    O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

    2015-01-01

    Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

  19. R-loops regulate promoter-proximal chromatin architecture and cellular differentiation

    PubMed Central

    Chen, Poshen B.; Chen, Hsiuyi V.; Acharya, Diwash; Rando, Oliver J.; Fazzio, Thomas G.

    2015-01-01

    Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R-loops, RNA:DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin regulatory complexes, Tip60–p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60–p400 histone acetyltransferase complex binds to nascent transcripts, but unlike PRC2, transcription promotes chromatin binding by Tip60–p400. Interestingly, we observed higher Tip60–p400 and lower PRC2 levels at genes marked by promoter-proximal R-loops. Furthermore, disruption of R-loops broadly reduced Tip60–p400 and increased PRC2 occupancy genome-wide. Consistent with these alterations, ESCs with reduced R-loops exhibited impaired differentiation. These results show that R-loops act both positively and negatively to modulate the recruitment of key pluripotency regulators. PMID:26551076

  20. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    PubMed

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division. PMID:24981860

  1. CTCF-mediated Chromatin Loop for the Posterior Hoxc Gene Expression in MEF Cells.

    PubMed

    Min, Hyehyun; Kong, Kyoung-Ah; Lee, Ji-Yeon; Hong, Chang-Pyo; Seo, Seong-Hye; Roh, Tae-Young; Bae, Sun Sik; Kim, Myoung Hee

    2016-06-01

    Modulation of chromatin structure has been proposed as a molecular mechanism underlying the spatiotemporal collinear expression of Hox genes during development. CCCTC-binding factor (CTCF)-mediated chromatin organization is now recognized as a crucial epigenetic mechanism for transcriptional regulation. Thus, we examined whether CTCF-mediated chromosomal conformation is involved in Hoxc gene expression by comparing wild-type mouse embryonic fibroblast (MEF) cells expressing anterior Hoxc genes with Akt1 null MEFs expressing anterior as well as posterior Hoxc genes. We found that CTCF binding between Hoxc11 and -c12 is important for CTCF-mediated chromosomal loop formation and concomitant posterior Hoxc gene expression. Hypomethylation at this site increased CTCF binding and recapitulated the chromosomal conformation and posterior Hoxc gene expression patterns observed in Akt1 null MEFs. From this work we found that CTCF at the C12|11 does not function as a barrier/boundary, instead let the posterior Hoxc genes switch their interaction from inactive centromeric to active telomeric genomic niche, and concomitant posterior Hoxc gene expression. Although it is not clear whether CTCF affects Hoxc gene expression solely through its looping activity, CTCF-mediated chromatin structural modulation could be an another tier of Hox gene regulation during development. © 2016 IUBMB Life, 68(6):436-444, 2016. PMID:27080371

  2. R loops regulate promoter-proximal chromatin architecture and cellular differentiation.

    PubMed

    Chen, Poshen B; Chen, Hsiuyi V; Acharya, Diwash; Rando, Oliver J; Fazzio, Thomas G

    2015-12-01

    Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R loops, RNA-DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin-regulatory complexes, Tip60-p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60-p400 histone acetyltransferase complex binds to nascent transcripts; however, transcription promotes chromatin binding of Tip60-p400 but not PRC2. Interestingly, we observed higher Tip60-p400 and lower PRC2 levels at genes marked by promoter-proximal R loops. Furthermore, disruption of R loops broadly decreased Tip60-p400 occupancy and increased PRC2 occupancy genome wide. In agreement with these alterations, ESCs partially depleted of R loops exhibited impaired differentiation. These results show that R loops act both positively and negatively in modulating the recruitment of key pluripotency regulators. PMID:26551076

  3. Sedimentary condensation and authigenesis

    NASA Astrophysics Data System (ADS)

    Föllmi, Karl

    2016-04-01

    Most marine authigenic minerals form in sediments, which are subjected to condensation. Condensation processes lead to the formation of well individualized, extremely thin (< 1m) beds, which were accumulated during extremely long time periods (> 100ky), and which experienced authigenesis and the precipitation of glaucony, verdine, phosphate, iron and manganese oxyhydroxides, iron sulfide, carbonate and/or silica. They usually show complex internal stratigraphies, which result from an interplay of sediment accumulation, halts in sedimentation, sediment winnowing, erosion, reworking and bypass. They may include amalgamated faunas of different origin and age. Hardgrounds may be part of condensed beds and may embody strongly condensed beds by themselves. Sedimentary condensation is the result of a hydrodynamically active depositional regime, in which sediment accumulation, winnowing, erosion, reworking and bypass are processes, which alternate as a function of changes in the location and intensity of currents, and/or as the result of episodic high-energy events engendered by storms and gravity flow. Sedimentary condensation has been and still is a widespread phenomenon in past and present-day oceans. The present-day distribution of glaucony and verdine-rich sediments on shelves and upper slopes, phosphate-rich sediments and phosphorite on outer shelves and upper slopes, ferromanganese crusts on slopes, seamounts and submarine plateaus, and ferromanganese nodules on abyssal seafloors is a good indication of the importance of condensation processes today. In the past, we may add the occurrence of oolitic ironstone, carbonate hardgrounds, and eventually also silica layers in banded iron formations as indicators of the importance of condensation processes. Besides their economic value, condensed sediments are useful both as a carrier of geochemical proxies of paleoceanographic and paleoenvironmental change, as well as the product of episodes of paleoceanographic and

  4. Electrolyte vapor condenser

    DOEpatents

    Sederquist, Richard A.; Szydlowski, Donald F.; Sawyer, Richard D.

    1983-01-01

    A system is disclosed for removing electrolyte from a fuel cell gas stream. The gas stream containing electrolyte vapor is supercooled utilizing conventional heat exchangers and the thus supercooled gas stream is passed over high surface area passive condensers. The condensed electrolyte is then drained from the condenser and the remainder of the gas stream passed on. The system is particularly useful for electrolytes such as phosphoric acid and molten carbonate, but can be used for other electrolyte cells and simple vapor separation as well.

  5. Electrolyte vapor condenser

    DOEpatents

    Sederquist, R.A.; Szydlowski, D.F.; Sawyer, R.D.

    1983-02-08

    A system is disclosed for removing electrolyte from a fuel cell gas stream. The gas stream containing electrolyte vapor is supercooled utilizing conventional heat exchangers and the thus supercooled gas stream is passed over high surface area passive condensers. The condensed electrolyte is then drained from the condenser and the remainder of the gas stream passed on. The system is particularly useful for electrolytes such as phosphoric acid and molten carbonate, but can be used for other electrolyte cells and simple vapor separation as well. 3 figs.

  6. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  7. Role of histone modifications in defining chromatin structure and function.

    PubMed

    Gelato, Kathy A; Fischle, Wolfgang

    2008-04-01

    Chromosomes in eukaryotic cell nuclei are not uniformly organized, but rather contain distinct chromatin elements, with each state having a defined biochemical structure and biological function. These are recognizable by their distinct architectures and molecular components, which can change in response to cellular stimuli or metabolic requirements. Chromatin elements are characterized by the fundamental histone and DNA components, as well as other associated non-histone proteins and factors. Post-translational modifications of histone proteins in particular often correlate with a specific chromatin structure and function. Patterns of histone modifications are implicated as having a role in directing the level of chromatin compaction, as well as playing roles in multiple functional pathways directing the readout of distinct regions of the genome. We review the properties of various chromatin elements and the apparent links of histone modifications with chromatin organization and functional output. PMID:18225984

  8. Chromatin dynamics associated with HIV-1 Tat activated transcription

    PubMed Central

    Easley, Rebecca; Van Duyne, Rachel; Coley, Will; Guendel, Irene; Dadgar, Sherry; Kehn-Hall, Kylene; Kashanchi, Fatah

    2009-01-01

    Summary Chromatin remodeling is an essential event for HIV-1 transcription. Over the last two decades this field of research has come to the forefront, as silencing of the HIV-1 provirus through chromatin modifications has been linked to latency. Here, we focus on chromatin remodeling, especially in relation to the transactivator Tat, and review the most important and newly emerging studies that investigate remodeling mechanisms. We begin by discussing covalent modifications that can alter chromatin structure including acetylation, deacetylation, and methylation, as well as topics addressing the interplay between chromatin remodeling and splicing. Next, we focus on complexes that use the energy of ATP to remove or secure nucleosomes and can additionally act to control HIV-1 transcription. Finally, we cover recent literature on viral microRNAs which have been shown to alter chromatin structure by inducing methylation or even by remodeling nucleosomes. PMID:19716452

  9. In Brief: Picturing the complex world of chromatin remodelling families.

    PubMed

    Witkowski, Leora; Foulkes, William D

    2015-12-01

    Over the past decade, chromatin remodelling emerged as one of the most important causes of both abnormal development and cancer. Although much has been written about one or another of the complexes, no recent concise summary of the chromatin remodelling families as a whole is available. In this short review, we introduce the family members, briefly summarize their role in developmental abnormalities and neoplasia, and outline the different ways in which these families remodel chromatin. PMID:26174723

  10. 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.

  11. Setd1a and NURF mediate chromatin dynamics and gene regulation during erythroid lineage commitment and differentiation

    PubMed Central

    Li, Ying; Schulz, Vincent P.; Deng, Changwang; Li, Guangyao; Shen, Yong; Tusi, Betsabeh K.; Ma, Gina; Stees, Jared; Qiu, Yi; Steiner, Laurie A.; Zhou, Lei; Zhao, Keji; Bungert, Jörg; Gallagher, Patrick G.; Huang, Suming

    2016-01-01

    The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult β-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation. PMID:27141965

  12. Stem cell factors in plants: chromatin connections.

    PubMed

    Kornet, N; Scheres, B

    2008-01-01

    The progression of pluripotent stem cells to differentiated cell lineages requires major shifts in cell differentiation programs. In both mammals and higher plants, this process appears to be controlled by a dedicated set of transcription factors, many of which are kingdom specific. These divergent transcription factors appear to operate, however, together with a shared suite of factors that affect the chromatin state. It is of major importance to investigate whether such shared global control mechanisms indicate a common mechanistic basis for preservation of the stem cell state, initiation of differentiation programs, and coordination of cell state transitions. PMID:19150963

  13. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    PubMed Central

    Bushey, Ashley M.; Dorman, Elizabeth R.; Corces, Victor G.

    2008-01-01

    Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function. PMID:18851828

  14. Hydrogen peroxide mediates higher order chromatin degradation.

    PubMed

    Bai, H; Konat, G W

    2003-01-01

    Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2). PMID:12421592

  15. Ghost condensate busting

    SciTech Connect

    Bilic, Neven; Tupper, Gary B; Viollier, Raoul D E-mail: gary.tupper@uct.ac.za

    2008-09-15

    Applying the Thomas-Fermi approximation to renormalizable field theories, we construct ghost condensation models that are free of the instabilities associated with violations of the null-energy condition.

  16. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

    PubMed Central

    2014-01-01

    Background Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Results We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Conclusions Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility. PMID:24581456

  17. The BAF60 subunit of the SWI/SNF chromatin-remodeling complex directly controls the formation of a gene loop at FLOWERING LOCUS C in Arabidopsis.

    PubMed

    Jégu, Teddy; Latrasse, David; Delarue, Marianne; Hirt, Heribert; Domenichini, Séverine; Ariel, Federico; Crespi, Martin; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2014-02-01

    SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus. PMID:24510722

  18. Measure Guideline: Evaporative Condensers

    SciTech Connect

    German, A.; Dakin, B.; Hoeschele, M.

    2012-03-01

    The purpose of this measure guideline on evaporative condensers is to provide information on a cost-effective solution for energy and demand savings in homes with cooling loads. This is a prescriptive approach that outlines selection criteria, design and installation procedures, and operation and maintenance best practices. This document has been prepared to provide a process for properly designing, installing, and maintaining evaporative condenser systems as well as understanding the benefits, costs, and tradeoffs.

  19. The Centromere: Chromatin Foundation for the Kinetochore Machinery

    PubMed Central

    Fukagawa, Tatsuo; Earnshaw, William C.

    2014-01-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

  20. The centromere: chromatin foundation for the kinetochore machinery.

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

  1. Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

    PubMed Central

    Eichten, Steven R.; Schmitz, Robert J.; Springer, Nathan M.

    2014-01-01

    Chromatin modifications and epigenetics may play important roles in many plant processes, including developmental regulation, responses to environmental stimuli, and local adaptation. Chromatin modifications describe biochemical changes to chromatin state, such as alterations in the specific type or placement of histones, modifications of DNA or histones, or changes in the specific proteins or RNAs that associate with a genomic region. The term epigenetic is often used to describe a variety of unexpected patterns of gene regulation or inheritance. Here, we specifically define epigenetics to include the key aspects of heritability (stable transmission of gene expression states through mitotic or meiotic cell divisions) and independence from DNA sequence changes. We argue against generically equating chromatin and epigenetics; although many examples of epigenetics involve chromatin changes, those chromatin changes are not always heritable or may be influenced by genetic changes. Careful use of the terms chromatin modifications and epigenetics can help separate the biochemical mechanisms of regulation from the inheritance patterns of altered chromatin states. Here, we also highlight examples in which chromatin modifications and epigenetics affect important plant processes. PMID:24872382

  2. Pioneer transcription factors, chromatin dynamics, and cell fate control.

    PubMed

    Zaret, Kenneth S; Mango, Susan E

    2016-04-01

    Among the diverse transcription factors that are necessary to elicit changes in cell fate, both in embryonic development and in cellular reprogramming, a subset of factors are capable of binding to their target sequences on nucleosomal DNA and initiating regulatory events in silent chromatin. Such 'pioneer transcription factors' initiate cooperative interactions with other regulatory proteins to elicit changes in local chromatin structure. As a consequence of pioneer factor binding, the local chromatin can either become open and competent for activation, closed and repressed, or transcriptionally active. Understanding how pioneer factors initiate chromatin dynamics and how such can be blocked at heterochromatic sites provides insights into controlling cell fate transitions at will. PMID:26826681

  3. Formaldehyde crosslinking: a tool for the study of chromatin complexes.

    PubMed

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

    2015-10-30

    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

  4. Upgrading the GSI beamline microscope with a confocal fluorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells

    NASA Astrophysics Data System (ADS)

    Abdollahi, Elham; Taucher-Scholz, Gisela; Durante, Marco; Jakob, Burkhard

    2015-12-01

    We report the upgrade of the GSI beamline microscope coupled to the linear accelerator UNILAC by a confocal FLIM scanner utilizing time correlated single photon counting technique (TCSPC). The system can now be used to address the radiation induced chromatin decondensation in more detail and with higher sensitivity compared to intensity based methods. This decondensation of heterochromatic areas is one of the early DNA damage responses observed after charged particle irradiation and might facilitate the further processing of the induced lesions. We describe here the establishment of different DNA dyes as chromatin compaction probes usable for quantification of the DNA condensation status in living cells utilizing lifetime imaging. In addition, we find an evidence of heterochromatic chromatin decondensation in ion irradiated murine chromocenters detected after subsequent fixation using FLIM measurements.

  5. The Fun30 Chromatin Remodeler Fft3 Controls Nuclear Organization and Chromatin Structure of Insulators and Subtelomeres in Fission Yeast

    PubMed Central

    Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-01-01

    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. PMID:25798942

  6. 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.

  7. Condensate dark matter stars

    SciTech Connect

    Li, X.Y.; Harko, T.; Cheng, K.S. E-mail: harko@hkucc.hku.hk

    2012-06-01

    We investigate the structure and stability properties of compact astrophysical objects that may be formed from the Bose-Einstein condensation of dark matter. Once the critical temperature of a boson gas is less than the critical temperature, a Bose-Einstein Condensation process can always take place during the cosmic history of the universe. Therefore we model the dark matter inside the star as a Bose-Einstein condensate. In the condensate dark matter star model, the dark matter equation of state can be described by a polytropic equation of state, with polytropic index equal to one. We derive the basic general relativistic equations describing the equilibrium structure of the condensate dark matter star with spherically symmetric static geometry. The structure equations of the condensate dark matter stars are studied numerically. The critical mass and radius of the dark matter star are given by M{sub crit} ≈ 2(l{sub a}/1fm){sup 1/2}(m{sub χ}/1 GeV){sup −3/2}M{sub s}un and R{sub crit} ≈ 1.1 × 10{sup 6}(l{sub a}/1 fm){sup 1/2}(m{sub χ}/1 GeV){sup −3/2} cm respectively, where l{sub a} and m{sub χ} are the scattering length and the mass of dark matter particle, respectively.

  8. Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin*

    PubMed Central

    Hendrix, Jelle; van Heertum, Bart; Vanstreels, Els; Daelemans, Dirk; De Rijck, Jan

    2014-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins. PMID:24634210

  9. ARTEMIS nuclease facilitates apoptotic chromatin cleavage.

    PubMed

    Britton, Sébastien; Frit, Philippe; Biard, Denis; Salles, Bernard; Calsou, Patrick

    2009-10-15

    One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis. PMID:19808974

  10. Histone modification and chromatin remodeling during NER.

    PubMed

    Waters, Raymond; van Eijk, Patrick; Reed, Simon

    2015-12-01

    Here we review our developments of and results with high resolution studies on global genome nucleotide excision repair (GG-NER) in Saccharomyces cerevisiae. Technologies were developed to examine NER at nucleotide resolution in yeast sequences of choice and to determine how these related to local changes in chromatin. We focused on how GG-NER relates to histone acetylation for its functioning and we identified the histone acetyltransferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. Factors influencing this Gcn5-mediated event are considered which include Rad16, a GG-NER specific SWI/SNF factor and the yeast histone variant of H2AZ (Htz1). We describe results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then consider the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the NER of UV-induced cyclobutane pyrimidine dimers throughout entire yeast genome. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage before it is returned to its pre-damaged status to maintain epigenetic codes. PMID:26422133

  11. Facioscapulohumeral muscular dystrophy: consequences of chromatin relaxation

    PubMed Central

    van der Maarel, Silvère M.; Miller, Daniel G.; Tawil, Rabi; Filippova, Galina N.; Tapscott, Stephen J.

    2013-01-01

    Purpose of review In recent years we have seen remarkable progress in our understanding of the disease mechanism underlying facioscapulohumeral muscular dystrophy (FSHD). The purpose of this review is to provide a comprehensive overview of our current understanding of the disease mechanism and to discuss the observations supporting the possibility of a developmental defect in this disorder. Recent findings In the majority of cases FSHD is caused by contraction of the D4Z4 repeat array (FSHD1). This results in local chromatin relaxation and stable expression of the DUX4 retrogene in skeletal muscle, but only when a polymorphic DUX4 polyadenylation signal is present. In some cases (FSHD2), D4Z4 chromatin relaxation and stable DUX4 expression occurs in the absence of D4Z4 array contraction. DUX4 is a germline transcription factor and its expression in skeletal muscle leads to activation of early stem cell and germline programs and transcriptional activation of retroelements. Summary Recent studies have provided a plausible disease mechanism for FSHD where FSHD results from inappropriate expression of the germline transcription factor DUX4. The genes regulated by DUX4 suggest several mechanisms of muscle damage, and provide potential biomarkers and therapeutic targets that should be investigated in future studies. PMID:22892954

  12. 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

  13. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

    PubMed Central

    Ray, Poulomi; Chapman, Susan C.

    2015-01-01

    Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

  14. The difference in LET and ion species dependence for induction of initially measured and non-rejoined chromatin breaks in normal human fibroblasts.

    PubMed

    Tsuruoka, Chizuru; Suzuki, Masao; Hande, M Prakash; Furusawa, Yoshiya; Anzai, Kazunori; Okayasu, Ryuichi

    2008-08-01

    We studied the LET and ion species dependence of the induction of chromatin breaks measured immediately after irradiation as initially measured breaks and after 24 h postirradiation incubation (37 degrees C) as non-rejoined breaks in normal human fibroblasts with different heavy ions, such as carbon, neon, silicon and iron, generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS). Chromatin breaks were measured as an excess number of fragments of prematurely condensed chromosomes using premature chromosome condensation (PCC). The results showed that the number of excess fragments per cell per Gy for initially measured chromatin breaks was dependent on LET in the range from 13.3 to 113.1 keV/mum but was not dependent on ion species. On the other hand, the number of non-rejoined chromatin breaks detected after 24 h postirradiation incubation was clearly dependent on both LET and ion species. No significant difference was observed in the cross section for initially measured breaks, but a statistically significant difference was observed in the cross section for non-rejoined breaks among carbon, neon, silicon and iron ions. This suggests that the LET-dependent structure in the biological effects is reflected in biological consequences of repair processes. PMID:18666815

  15. [Experimental visualization of chromoneme as one of the higher levels of chromatin compactization in the mitotic chromosome].

    PubMed

    Burakov, V V; Tvorogova, A V; Chentsov, Iu S

    2005-01-01

    We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution

  16. Chromatin regulates origin activity in Drosophila follicle cells.

    PubMed

    Aggarwal, Bhagwan D; Calvi, Brian R

    2004-07-15

    It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa. PMID:15254542

  17. An Oestrogen Receptor α-bound Human Chromatin Interactome

    PubMed Central

    Fullwood, Melissa J.; Liu, Mei Hui; Pan, You Fu; Liu, Jun; Han, Xu; Mohamed, Yusoff Bin; Orlov, Yuriy L.; Velkov, Stoyan; Ho, Andrea; Mei, Poh Huay; Chew, Elaine G. Y.; Huang, Phillips Yao Hui; Welboren, Willem-Jan; Han, Yuyuan; Ooi, Hong-Sain; Ariyaratne, Pramila N.; Vega, Vinsensius B.; Luo, Yanquan; Tan, Peck Yean; Choy, Pei Ye; Wansa, K. D. Senali Abayratna; Zhao, Bing; Lim, Kar Sian; Leow, Shi Chi; Yow, Jit Sin; Joseph, Roy; Li, Haixia; Desai, Kartiki V.; Thomsen, Jane S.; Lee, Yew Kok; Karuturi, R. Krishna Murthy; Herve, Thoreau; Bourque, Guillaume; Stunnenberg, Hendrik G.; Ruan, Xiaoan; Cacheux-Rataboul, Valere; Sung, Wing-Kin; Liu, Edison T.; Wei, Chia-Lin; Cheung, Edwin; Ruan, Yijun

    2009-01-01

    Genomes are organized into high-level 3-dimensional structures, and DNA elements separated by long genomic distances could functionally interact. Many transcription factors bind to regulatory DNA elements distant from gene promoters. While distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Therefore, we developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ERα) in the human genome. We found that most high-confidence remote ERα binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ERα functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes. PMID:19890323

  18. Chromatin Regulators as a Guide for Cancer Treatment Choice.

    PubMed

    Gurard-Levin, Zachary A; Wilson, Laurence O W; Pancaldi, Vera; Postel-Vinay, Sophie; Sousa, Fabricio G; Reyes, Cecile; Marangoni, Elisabetta; Gentien, David; Valencia, Alfonso; Pommier, Yves; Cottu, Paul; Almouzni, Geneviève

    2016-07-01

    The limited capacity to predict a patient's response to distinct chemotherapeutic agents is a major hurdle in cancer management. The efficiency of a large fraction of current cancer therapeutics (radio- and chemotherapies) is influenced by chromatin structure. Reciprocally, alterations in chromatin organization may affect resistance mechanisms. Here, we explore how the misexpression of chromatin regulators-factors involved in the establishment and maintenance of functional chromatin domains-can inform about the extent of docetaxel response. We exploit Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft models and patient samples treated with docetaxel. Random Forest classification reveals specific panels of chromatin regulators, including key components of the SWI/SNF chromatin remodeler, which readily distinguish docetaxel high-responders and poor-responders. Further exploration of SWI/SNF components in the comprehensive NCI-60 dataset reveals that the expression inversely correlates with docetaxel sensitivity. Finally, we show that loss of the SWI/SNF subunit BRG1 (SMARCA4) in a model cell line leads to enhanced docetaxel sensitivity. Altogether, our findings point toward chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients toward docetaxel and combat drug resistance. Mol Cancer Ther; 15(7); 1768-77. ©2016 AACR. PMID:27196757

  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. PMID:26381410

  20. 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.

  1. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    Donà, Mattia; Mittelsten Scheid, Ortrun

    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

  2. DNA Damage Repair in the Context of Plant Chromatin.

    PubMed

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-08-01

    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

  3. Data on the kinetics of in vitro assembled chromatin.

    PubMed

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

    2016-09-01

    Here, we use LC-MS/MS and SWATH-MS to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos (DREX). This system allows easy manipulation of distinct aspects of chromatin assembly such as post-translational histone modifications, the levels of histone chaperones and the concentration of distinct DNA binding factors. In total, 480 proteins have been quantified as chromatin enriched factors and their binding kinetics have been monitored in the time course of 15 min, 1 h and 4 h of chromatin assembly. The data accompanying the manuscript on this approach, Völker-Albert et al., 2016 "A quantitative proteomic analysis of in vitro assembled chromatin" [1], has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier submission number PRIDE: PXD002537 and PRIDE: PXD003445. PMID:27331114

  4. Higher order chromatin structure: bridging physics and biology

    PubMed Central

    Fudenberg, Geoffrey; Mirny, Leonid A.

    2012-01-01

    Recent advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of high-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently-developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization. PMID:22360992

  5. Chromatin and epigenetic features of long-range gene regulation

    PubMed Central

    Harmston, Nathan; Lenhard, Boris

    2013-01-01

    The precise regulation of gene transcription during metazoan development is controlled by a complex system of interactions between transcription factors, histone modifications and modifying enzymes and chromatin conformation. Developments in chromosome conformation capture technologies have revealed that interactions between regions of chromatin are pervasive and highly cell-type specific. The movement of enhancers and promoters in and out of higher-order chromatin structures within the nucleus are associated with changes in expression and histone modifications. However, the factors responsible for mediating these changes and determining enhancer:promoter specificity are still not completely known. In this review, we summarize what is known about the patterns of epigenetic and chromatin features characteristic of elements involved in long-range interactions. In addition, we review the insights into both local and global patterns of chromatin interactions that have been revealed by the latest experimental and computational methods. PMID:23766291

  6. Enhanced condensation heat transfer

    NASA Astrophysics Data System (ADS)

    Michel, J. W.; Murphy, R. W.

    1980-07-01

    Work has centered on optimizing the design variables associated with fluted surfaces on vertical tubes and comparing the tube performance with available enhanced tubes either for vertical or horizontal operation. Data with seven fluids including a hydrocarbon, fluorocarbons, and ammonia condensing on up to 30 different tubes were obtained. Data for tubes of different effective lengths (1/2 to 4 ft) and inclination were also obtained. The primary conclusion is that the best fluted tubes can provide an enhancement in condensation coefficient by a factor of approximately 6 over smooth vertical tube performance and a factor of approximately 2 over the best enhanced commercial tubes either operating vertically or horizontally. These data, together with field test data, have formed the basis for designing two prototype condensers, one for the 60 kWe Raft River, Idaho, pilot plant and one for the 500 kWe East Mesa, California, direct contact demonstration plant.

  7. Chromatin organisation in duckweed interphase nuclei in relation to the nuclear DNA content.

    PubMed

    Cao, H X; Vu, G T H; Wang, W; Messing, J; Schubert, I

    2015-01-01

    The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus. PMID:24853858

  8. Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function.

    PubMed

    Seol, Ja-Hwan; Song, Tae-Yang; Oh, Se Eun; Jo, Chanhee; Choi, Ahreum; Kim, Byungho; Park, Jinyoung; Hong, Suji; Song, Ilrang; Jung, Kwan Young; Yang, Jae-Hyun; Park, Hwangseo; Ahn, Jin-Hyun; Han, Jeung-Whan; Cho, Eun-Jung

    2015-12-01

    The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modification. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. PMID:26058396

  9. Myc-binding-site recognition in the human genome is determined by chromatin context.

    PubMed

    Guccione, Ernesto; Martinato, Francesca; Finocchiaro, Giacomo; Luzi, Lucilla; Tizzoni, Laura; Dall' Olio, Valentina; Zardo, Giuseppe; Nervi, Clara; Bernard, Loris; Amati, Bruno

    2006-07-01

    Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo. PMID:16767079

  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. Identification of alternative topological domains in chromatin

    PubMed Central

    2014-01-01

    Chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The ensemble of domains we identify allows us to quantify the degree to which the domain structure is hierarchical as opposed to overlapping, and our analysis reveals a pronounced hierarchical structure in which larger stable domains tend to completely contain smaller domains. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone marks at the boundaries. PMID:24868242

  12. Properties of intracellular bovine papillomavirus chromatin.

    PubMed Central

    Rösl, F; Waldeck, W; Zentgraf, H; Sauer, G

    1986-01-01

    Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence. Images PMID:3009863

  13. Effect of DNA Groove Binder Distamycin A upon Chromatin Structure

    PubMed Central

    Majumder, Parijat; Dasgupta, Dipak

    2011-01-01

    Background Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. Methodology/Principal Findings Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods - dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. Conclusions/Significance We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture

  14. Keeping condensers clean

    SciTech Connect

    Wicker, K.

    2006-04-15

    The humble condenser is among the biggest contributors to a steam power plant's efficiency. But although a clean condenser can provide great economic benefit, a dirty one can raise plant heat rate, resulting in large losses of generation revenue and/or unnecessarily high fuel bills. Conventional methods for cleaning fouled tubes range form chemicals to scrapers to brushes and hydro-blasters. This article compares the available options and describes how one power station, Omaha Public Power District's 600 MW North Omaha coal-fired power station, cleaned up its act. The makeup and cooling water of all its five units comes from the Missouri River. 6 figs.

  15. 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

  16. Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

    PubMed

    Hirakawa, Takeshi; Matsunaga, Sachihiro

    2016-01-01

    In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants. PMID:27557696

  17. Chromatin remodelling and histone m RNA accumulation in bovine germinal vesicle oocytes.

    PubMed

    Labrecque, R; Lodde, V; Dieci, C; Tessaro, I; Luciano, A M; Sirard, M A

    2015-06-01

    Major remodelling of the chromatin enclosed within the germinal vesicle occurs towards the end of oocyte growth in mammals, but the mechanisms involved in this process are not completely understood. In bovine, four distinct stages of chromatin compaction-ranging from a diffused state (GV0) to a fully compacted configuration (GV3)-are linked to the gradual acquisition of developmental potential. To better understand the molecular events and to identify mRNA modulations occurring in the oocyte during the GV0-to-GV3 transition, transcriptomic analysis was performed with the EmbryoGENE microarray platform. The mRNA abundance of several genes decreased as chromatin compaction increased, which correlates with progressive transcriptional silencing that is characteristic of the end of oocyte growth. On the other hand, the abundance of some transcripts increased during the same period, particularly several histone gene transcripts from the H2A, H2B, H3, H4, and linker H1 family. In silico analysis predicted RNA-protein interactions between specific histone transcripts and the bovine stem-loop binding protein 2 (SLBP2), which helps regulate the translation of histone mRNA during oogenesis. These results suggest that some histone-encoding transcripts are actively stored, possibly to sustain the needs of the embryo before genome activation. This dataset offers a unique opportunity to survey which histone mRNAs are needed to complete chromatin compaction during oocyte maturation and which are stockpiled for the first three cell cycles following fertilization. PMID:25940597

  18. Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

    PubMed

    Sutcliffe, Elissa L; Li, Jasmine; Zafar, Anjum; Hardy, Kristine; Ghildyal, Reena; McCuaig, Robert; Norris, Nicole C; Lim, Pek Siew; Milburn, Peter J; Casarotto, Marco G; Denyer, Gareth; Rao, Sudha

    2012-01-01

    We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer. PMID:22969762

  19. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put

    PubMed Central

    Simon, Jeffrey A.; Kingston, Robert E.

    2013-01-01

    Summary Polycomb repressive complexes are conserved chromatin regulators with key roles in multicellular development, stem cell biology, and cancer. New findings advance molecular understanding of how they target to sites of action, interact with and alter local chromatin to silence genes, and maintain silencing in successive generations of proliferating cells. Chromatin modification by Polycomb proteins provides an essential strategy for gene silencing in higher eukaryotes. Polycomb repressive complexes (PRCs) silence many key developmental regulators and are centrally integrated in the transcriptional circuitry of embryonic and adult stem cells. PRC2 trimethylates histone H3 on lysine-27 (H3-K27me3) and PRC1-type complexes ubiquitylate histone H2A and compact polynucleosomes. How PRCs and these signature activities are deployed to select and silence genomic targets is the subject of intense current investigation. We review recent advances on targeting, modulation, and functions of PRC1 and PRC2, and we consider progress on defining transcriptional steps impacted in Polycomb silencing. Key recent findings demonstrate PRC1 targeting independent of H3-K27me3 and emphasize nonenzymatic PRC1-mediated compaction. We also evaluate expanding connections between Polycomb machinery and non-coding RNAs. Exciting new studies supply the first systematic analyses of what happens to Polycomb complexes, and associated histone modifications, during the wholesale chromatin reorganizations that accompany DNA replication and mitosis. The stage is now set to reveal fundamental epigenetic mechanisms that determine how Polycomb target genes are silenced and how Polycomb silence is preserved through cell cycle progression. PMID:23473600

  20. Possible role of chromatin alteration in the radiosensitivity of ataxia-telangiectasia.

    PubMed

    Hittelman, W N; Pandita, T K

    1994-12-01

    Cells derived from individuals with ataxia-telangiectasia (A-T) are known to exhibit increased sensitivity to ionizing radiation and certain radiomimetic chemical agents. Here we summarize our findings regarding the role of chromosome damage and repair in this radiosensitivity. Lymphoblastoid cells derived from A-T homozygotes were characterized for initial chromosome (premature chromosome condensation) and DNA (neutral filter elution) damage and repair kinetics in cells from G1 and G2 cell cycle phases. Despite initial levels of DNA damage being similar to normal controls, A-T cells exhibited nearly a two-fold higher initial amount of chromosome damage. Different A-T cell lines exhibited differing chromosome repair capacities compared with control lymphoblastoid cell lines. These results suggest that A-T cells have an altered chromatin structure whereby DNA double-strand breaks are apparently more efficiently converted into chromosome breaks. Four A-T heterozygote cell lines were examined for chromosome damage and repair in the same fashion and all exhibited increased levels of chromosome damage, although the degree of sensitivity was more prominent in G2 phase cells (two-fold higher) than in G1 phase cells (1.5-fold higher than normal controls). These results suggest that A-T heterozygotes also exhibit an altered chromatin structure which impacts on chromosome damage expression. Of interest, A-T cells also exhibited increased chromosome stickiness after irradiation, and telomere regions appeared to be frequently involved. While the molecular basis for preferential telomere involvement is not understood, these results again suggest that structural alterations in the chromatin of A-T cells may play an important role in A-T radiosensitivity. PMID:7836837

  1. Simple Simulations of DNA Condensation

    SciTech Connect

    STEVENS,MARK J.

    2000-07-12

    Molecular dynamics simulations of a simple, bead-spring model of semiflexible polyelectrolytes such as DNA are performed. All charges are explicitly treated. Starting from extended, noncondensed conformations, condensed structures form in the simulations with tetravalent or trivalent counterions. No condensates form or are stable for divalent counterions. The mechanism by which condensates form is described. Briefly, condensation occurs because electrostatic interactions dominate entropy, and the favored Coulombic structure is a charge ordered state. Condensation is a generic phenomena and occurs for a variety of polyelectrolyte parameters. Toroids and rods are the condensate structures. Toroids form preferentially when the molecular stiffness is sufficiently strong.

  2. Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin.

    PubMed

    Caplan, A; Kimura, T; Gould, H; Allan, J

    1987-01-01

    An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour. PMID:3586025

  3. Detail of Bright Angel stone vault, containing condenser, Hoffman condensation ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail of Bright Angel stone vault, containing condenser, Hoffman condensation pump, Jennings vacuum heating pump, and misc. pipes and valves. - Grand Canyon Village Utilities, Grand Canyon National Park, Grand Canyon Village, Coconino County, AZ

  4. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling.

    PubMed

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G; Becker, Jörg D; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G

    2016-01-01

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I. PMID:27507044

  5. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

    PubMed Central

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G.; Becker, Jörg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G.

    2016-01-01

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I. PMID:27507044

  6. Evidence supporting a biological role for aluminum in brain chromatin compaction and epigenetics.

    PubMed

    Lukiw, Walter J

    2010-05-19

    Averaging 8.1% (w/w) of the earth's crust, aluminum is the most highly abundant metal in our biosphere, yet has long been thought to serve no essential biological function. In aqueous solutions, aluminum salts and hydroxides are exceptionally potent aggregators of biological molecules, often coalescing molecular species to the point that they precipitate out of solution. A biological function for aluminum is proposed in which this abundant, high charge density metal cation has a significant role in biomolecular compaction. Sometimes, molecules ectopically aggregated by aluminum are associated with pathological conditions. The data further suggests that a specific consequence of 'aluminum biocompaction' may be particularly important in the condensation of A+T-rich chromatin domains, and in silencing the expression of specific kinds of genetic information. PMID:20684847

  7. Inflation from gravitino condensates

    NASA Astrophysics Data System (ADS)

    Mavromatos, Nick E.

    2015-07-01

    We review work on the formation of gravitino condensates via the super-Higgs effect in the early Universe. This is a scenario for both inflating the early universe and breaking local supersymmetry(supergravity), entirely independent of any coupling to external matter. The goldstino mode associated with the breaking of (global) supersymmetry is “eaten” by the gravitino field, which becomes massive (via its own vacuum condensation) and breaks supergravity dynamically. The most natural association of gravitino condensates with inflation proceeds in an indirect way, via a Starobinsky-type inflation, in the massive gravitino phase. This inflationary phase is associated with scalar modes hidden in the higher order curvature corrections of the effective action arising from integrating out massive gravitino degrees of freedom. The scenario is in agreement with Planck data phenomenology in a natural and phenomenologically-relevant range of parameters, namely Grand-Unified-Theory values for the supersymmetry breaking energy scale and dynamically-induced gravitino mass. A hill-top inflation, on the other hand, which could also occur in the model, whereby the role of the inflaton field is played by the gravitino condensate itself, would require significant fine tuning in the inflaton's wave function renormalisation and thus may be discarded on naturalness grounds.

  8. Condensate removal device

    DOEpatents

    Maddox, James W.; Berger, David D.

    1984-01-01

    A condensate removal device is disclosed which incorporates a strainer in unit with an orifice. The strainer is cylindrical with its longitudinal axis transverse to that of the vapor conduit in which it is mounted. The orifice is positioned inside the strainer proximate the end which is remoter from the vapor conduit.

  9. Minireview: role of kinases and chromatin remodeling in progesterone signaling to chromatin.

    PubMed

    Vicent, Guillermo P; Nacht, A Silvina; Zaurín, Roser; Ballaré, Cecilia; Clausell, Jaime; Beato, Miguel

    2010-11-01

    Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsive elements on DNA or with other transcription factors, but they can also activate cytoplasmic signaling cascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR) with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus (MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylation of PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complex of the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome, resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein 1γ. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes. Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF) and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoter and cooperates with PCAF during activation of hormone-responsive promoters. PCAF acetylates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring the complex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facilitating access of nuclear factor 1 and additional PR complexes to the hidden hormone-responsive elements on the MMTV promoter. The linker histone H1 is a structural component of chromatin generally regarded as a general repressor of transcription. However, it contributes to a better regulation of the MMTV promoter by favoring a more homogeneous nucleosome positioning, thus reducing basal transcription and actually enhancing hormone induced transcription. During transcriptional activation, H1 is phosphorylated and displaced from the promoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment and could

  10. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

    PubMed Central

    Máthé, Csaba; M-Hamvas, Márta; Vasas, Gábor

    2013-01-01

    Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization. PMID:24084787

  11. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability

    PubMed Central

    Li, Lei; Shi, Lan; Yang, Shangda; Yan, Ruorong; Zhang, Di; Yang, Jianguo; He, Lin; Li, Wanjin; Yi, Xia; Sun, Luyang; Liang, Jing; Cheng, Zhongyi; Shi, Lei; Shang, Yongfeng; Yu, Wenhua

    2016-01-01

    Although SIRT7 is a member of sirtuin family proteins that are described as NAD+-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD+-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7. PMID:27436229

  12. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-01

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism. PMID:27108550

  13. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability.

    PubMed

    Li, Lei; Shi, Lan; Yang, Shangda; Yan, Ruorong; Zhang, Di; Yang, Jianguo; He, Lin; Li, Wanjin; Yi, Xia; Sun, Luyang; Liang, Jing; Cheng, Zhongyi; Shi, Lei; Shang, Yongfeng; Yu, Wenhua

    2016-01-01

    Although SIRT7 is a member of sirtuin family proteins that are described as NAD(+)-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD(+)-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7. PMID:27436229

  14. Transcriptional response to stress in the dynamic chromatin environment of cycling and mitotic cells

    PubMed Central

    Vihervaara, Anniina; Sergelius, Christian; Vasara, Jenni; Blom, Malin A. H.; Elsing, Alexandra N.; Roos-Mattjus, Pia; Sistonen, Lea

    2013-01-01

    Heat shock factors (HSFs) are the master regulators of transcription under protein-damaging conditions, acting in an environment where the overall transcription is silenced. We determined the genomewide transcriptional program that is rapidly provoked by HSF1 and HSF2 under acute stress in human cells. Our results revealed the molecular mechanisms that maintain cellular homeostasis, including HSF1-driven induction of polyubiquitin genes, as well as HSF1- and HSF2-mediated expression patterns of cochaperones, transcriptional regulators, and signaling molecules. We characterized the genomewide transcriptional response to stress also in mitotic cells where the chromatin is tightly compacted. We found a radically limited binding and transactivating capacity of HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In contrast, HSF2 occupied hundreds of loci in the mitotic cells and localized to the condensed chromatin also in meiosis. These results highlight the importance of the cell cycle phase in transcriptional responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 is an epigenetic regulator directing transcription throughout cell cycle progression. PMID:23959860

  15. 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

  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. PMID:27006400

  17. CTCF-Mediated Functional Chromatin Interactome in Pluripotent Cells

    PubMed Central

    Handoko, Lusy; Xu, Han; Li, Guoliang; Ngan, Chew Yee; Chew, Elaine; Schnapp, Marie; Lee, Charlie Wah Heng; Ye, Chaopeng; Ping, Joanne Lim Hui; Mulawadi, Fabianus; Wong, Eleanor; Sheng, Jianpeng; Zhang, Yubo; Poh, Thompson; Chan, Chee Seng; Kunarso, Galih; Shahab, Atif; Bourque, Guillaume; Cacheux-Rataboul, Valere; Sung, Wing-Kin; Ruan, Yijun; Wei, Chia-Lin

    2011-01-01

    Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function. PMID:21685913

  18. Chromatin decondensed by acetylation shows an elevated radiation response

    SciTech Connect

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-02-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.

  19. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  20. Mapping regulatory factors by immunoprecipitation from native chromatin

    PubMed Central

    Orsi, Guillermo A.; Kasinathan, Sivakanthan; Zentner, Gabriel E.; Henikoff, Steven; Ahmad, Kami

    2015-01-01

    Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this Unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors. PMID:25827087

  1. Chromatin regulation at the frontier of synthetic biology

    PubMed Central

    Keung, Albert J.; Joung, J. Keith; Khalil, Ahmad S.; Collins, James J.

    2016-01-01

    As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including `epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication. PMID:25668787

  2. Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures.

    PubMed

    Bignold, L P

    2002-01-01

    Nuclear chromatin patterns are used to distinguish normal and abnormal cells in histopathology and cytopathology. However, many chromatin pattern features are affected by aspects of tissue processing, especially fixation. Major effects of aldehyde and/or ethanol fixation on nuclei in the living state include shrinkage, chromatin aggregation and production of a 'chromatinic rim'. The mechanisms of these effects are poorly understood. In the past, possible mechanisms of fixation-induced morphological change have been considered only in terms of the theoretical model of the nucleus, which involves only a random tangle of partly unfolded chromosomes contained within the nuclear membrane. Such a model provides no basis for chromatin to be associated with the nuclear envelope, and hence no obvious clue to a mechanism for the formation of the 'chromatinic rim' in fixed nuclei. In recent years, two new models of nuclear structure have been described. The nuclear membrane-bound, chromosomal-domain model is based on the discoveries of chromatin-nuclear membrane attachments and of the localisation of the chromatin of each chromosome within discrete, exclusive parts of the nucleus (the 'domain' of each partly unfolded chromosome). The nuclear matrix/scaffold model is based on the discovery of relatively insoluble proteins in nuclei, which it suggests forms a 'matrix' and modulates gene expression by affecting transcription of DNA. Here, a hypothesis for fixation-associated chromatin pattern formation based mainly on the first model but partially relying on the second, is presented. The hypothesis offers explanations of the variations of appearance of nuclei according to fixation (especially air-drying versus wet-fixation with formaldehyde, glutaraldehyde or ethanol); the appearances of the nuclei of more metabolically active versus less metabolically active cells of the same type; the appearances of nuclei after fixation with osmium tetroxide; and of the marked central

  3. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.

    PubMed

    Franz, André; Pirson, Paul A; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

    2016-01-01

    The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

  4. Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

    PubMed Central

    MacAlpine, Heather K.; Lubelsky, Yoav; Hartemink, Alexander J.

    2015-01-01

    Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2–7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we “footprinted” nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2–7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). PMID:25593310

  5. Coordinated Regulation of TIP60 and Poly(ADP-Ribose) Polymerase 1 in Damaged-Chromatin Dynamics.

    PubMed

    Ikura, Masae; Furuya, Kanji; Fukuto, Atsuhiko; Matsuda, Ryo; Adachi, Jun; Matsuda, Tomonari; Kakizuka, Akira; Ikura, Tsuyoshi

    2016-05-15

    The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses. PMID:26976643

  6. ANGUSTIFOLIA3 Binds to SWI/SNF Chromatin Remodeling Complexes to Regulate Transcription during Arabidopsis Leaf Development[W

    PubMed Central

    Vercruyssen, Liesbeth; Verkest, Aurine; Gonzalez, Nathalie; Heyndrickx, Ken S.; Eeckhout, Dominique; Han, Soon-Ki; Jégu, Teddy; Archacki, Rafal; Van Leene, Jelle; Andriankaja, Megan; De Bodt, Stefanie; Abeel, Thomas; Coppens, Frederik; Dhondt, Stijn; De Milde, Liesbeth; Vermeersch, Mattias; Maleux, Katrien; Gevaert, Kris; Jerzmanowski, Andrzej; Benhamed, Moussa; Wagner, Doris; Vandepoele, Klaas; De Jaeger, Geert; Inzé, Dirk

    2014-01-01

    The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf. PMID:24443518

  7. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

    PubMed Central

    Franz, André; Pirson, Paul A.; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

    2016-01-01

    The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

  8. Epigenetic regulation by BAF (mSWI/SNF) chromatin remodeling complexes is indispensable for embryonic development.

    PubMed

    Nguyen, Huong; Sokpor, Godwin; Pham, Linh; Rosenbusch, Joachim; Stoykova, Anastassia; Staiger, Jochen F; Tuoc, Tran

    2016-05-18

    The multi-subunit chromatin-remodeling SWI/SNF (known as BAF for Brg/Brm-associated factor) complexes play essential roles in development. Studies have shown that the loss of individual BAF subunits often affects local chromatin structure and specific transcriptional programs. However, we do not fully understand how BAF complexes function in development because no animal mutant had been engineered to lack entire multi-subunit BAF complexes. Importantly, we recently reported that double conditional knock-out (dcKO) of the BAF155 and BAF170 core subunits in mice abolished the presence of the other BAF subunits in the developing cortex. The generated dcKO mutant provides a novel and powerful tool for investigating how entire BAF complexes affect cortical development. Using this model, we found that BAF complexes globally control the key heterochromatin marks, H3K27me2 and -3, by directly modulating the enzymatic activity of the H3K27 demethylases, Utx and Jmjd3. Here, we present further insights into how the scaffolding ability of the BAF155 and BAF170 core subunits maintains the stability of BAF complexes in the forebrain and throughout the embryo during development. Furthermore, we show that the loss of BAF complexes in the above-described model up-regulates H3K27me3 and impairs forebrain development and embryogenesis. These findings improve our understanding of epigenetic mechanisms and their modulation by the chromatin-remodeling SWI/SNF complexes that control embryonic development. PMID:26986003

  9. Sperm cryopreservation: effects on chromatin structure.

    PubMed

    Paoli, Donatella; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

    2014-01-01

    Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196°C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little - and contradictory - information on the mechanism of the generation of DNA fragmentation after cryopreservation. More studies are needed to establish the true importance of such damage, especially to improve the results of ART. PMID:23955677

  10. Chromatin and extracellular vesicle associated sperm RNAs

    PubMed Central

    Johnson, Graham D.; Mackie, Paula; Jodar, Meritxell; Moskovtsev, Sergey; Krawetz, Stephen A.

    2015-01-01

    A diverse pool of RNAs remain encapsulated within the transcriptionally silent spermatozoon despite the dramatic reduction in cellular and nuclear volume following cytoplasm/nucleoplasm expulsion. The impact of this pronounced restructuring on the distribution of transcripts inside the sperm essentially remains unknown. To define their compartmentalization, total RNA >100 nt was extracted from sonicated (SS) mouse spermatozoa and detergent demembranated sucrose gradient fractionated (Cs/Tx) sperm heads. Sperm RNAs predominately localized toward the periphery. The corresponding distribution of transcripts and thus localization and complexity were then inferred by RNA-seq. Interestingly, the number of annotated RNAs in the CsTx sperm heads exhibiting reduced peripheral enrichment was restricted. However this included Cabyr, the calcium-binding tyrosine phosphorylation-regulated protein encoded transcript. It is present in murine zygotes prior to the maternal to the zygotic transition yet absent in oocytes, consistent with the delivery of internally positioned sperm-borne RNAs to the embryo. In comparison, transcripts enriched in sonicated sperm contributed to the mitochondria and exosomes along with several nuclear transcripts including the metastasis associated lung adenocarcinoma transcript 1 (Malat1) and several small nucleolar RNAs. Their preferential peripheral localization suggests that chromatin remodeling during spermiogenesis is not limited to nucleoproteins as part of the nucleoprotein exchange. PMID:26071953

  11. Engineered apoptotic nucleases for chromatin research.

    PubMed

    Xiao, Fei; Widlak, Piotr; Garrard, William T

    2007-01-01

    We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5. PMID:17626049

  12. Multilayer graphene condenser microphone

    NASA Astrophysics Data System (ADS)

    Todorović, Dejan; Matković, Aleksandar; Milićević, Marijana; Jovanović, Djordje; Gajić, Radoš; Salom, Iva; Spasenović, Marko

    2015-12-01

    Vibrating membranes are the cornerstone of acoustic technology, forming the backbone of modern loudspeakers and microphones. Acoustic performance of a condenser microphone is derived mainly from the membrane’s size, surface mass and achievable static tension. The widely studied and available nickel has been a dominant membrane material for professional microphones for several decades. In this paper we introduce multilayer graphene as a membrane material for condenser microphones. The graphene device outperforms a high end commercial nickel-based microphone over a significant part of the audio spectrum, with a larger than 10 dB enhancement of sensitivity. Our experimental results are supported with numerical simulations, which also show that a 300 layer thick graphene membrane under maximum tension would offer excellent extension of the frequency range, up to 1 MHz.

  13. Gravitational vacuum condensate stars

    PubMed Central

    Mazur, Pawel O.; Mottola, Emil

    2004-01-01

    A new final state of gravitational collapse is proposed. By extending the concept of Bose–Einstein condensation to gravitational systems, a cold, dark, compact object with an interior de Sitter condensate pv = -ρv and an exterior Schwarzschild geometry of arbitrary total mass M is constructed. These regions are separated by a shell with a small but finite proper thickness ℓ of fluid with equation of state p = +ρ, replacing both the Schwarzschild and de Sitter classical horizons. The new solution has no singularities, no event horizons, and a global time. Its entropy is maximized under small fluctuations and is given by the standard hydrodynamic entropy of the thin shell, which is of the order kBℓMc/, instead of the Bekenstein–Hawking entropy formula, SBH = 4πkBGM2/c. Hence, unlike black holes, the new solution is thermodynamically stable and has no information paradox. PMID:15210982

  14. CW laser light condensation.

    PubMed

    Zhurahov, Michael; Bekker, Alexander; Levit, Boris; Weill, Rafi; Fischer, Baruch

    2016-03-21

    We present a first experimental demonstration of classical CW laser light condensation (LC) in the frequency (mode) domain that verifies its prediction (Fischer and Weill, Opt. Express20, 26704 (2012)). LC is based on weighting the modes in a noisy environment in a loss-gain measure compared to an energy (frequency) scale in Bose-Einstein condensation (BEC). It is characterized by a sharp transition from multi- to single-mode oscillation, occurring when the spectral-filtering (loss-trap) has near the lowest-loss mode ("ground-state") a power-law dependence with an exponent smaller than 1. An important meaning of the many-mode LC system stems from its relation to lasing and photon-BEC. PMID:27136845

  15. Bose-Einstein Condensation

    SciTech Connect

    El-Sherbini, Th.M.

    2005-03-17

    This article gives a brief review of Bose-Einstein condensation. It is an exotic quantum phenomenon that was observed in dilute atomic gases for the first time in 1995. It exhibits a new state of matter in which a group of atoms behaves as a single particle. Experiments on this form of matter are relevant to many different areas of physics- from atomic clocks and quantum computing to super fluidity, superconductivity and quantum phase transition.

  16. Insights into Chromatin Structure and Dynamics in Plants

    PubMed Central

    Rosa, Stefanie; Shaw, Peter

    2013-01-01

    The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

  17. Does seminal fluid viscosity influence sperm chromatin integrity?

    PubMed

    Gopalkrishnan, K; Padwal, V; Balaiah, D

    2000-01-01

    A retrospective study was undertaken to investigate whether viscosity alters sperm chromatin integrity. Semen samples were obtained from 269 men attending the infertility clinic. The viscosity was measured quantitatively by needle and syringe method and the viscosity ratio was calculated against distilled water. The chromatin integrity was evaluated by in vitro decondensation test using 1% SDS and 6 mM EDTA. According to the viscosity ratios the samples were divided into 2 groups: I, normal (ratio < 9, n = 239): and II, abnormal (ratio > 9, n = 30) viscosity. Chromatin integrity was significantly lower in the group with higher viscosity. Significant decrease in sperm count and motility were seen in group II as compared to group I. Thus, hyperviscosity of seminal fluid alters the sperm chromatin integrity. PMID:11028927

  18. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

    PubMed

    Bandaria, Jigar N; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet

    2016-02-11

    Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633

  19. Computational analysis of promoter elements and chromatin features in yeast.

    PubMed

    Wyrick, John J

    2012-01-01

    Regulatory elements in promoter sequences typically function as binding sites for transcription factor proteins and thus are critical determinants of gene transcription. There is growing evidence that chromatin features, such as histone modifications or nucleosome positions, also have important roles in transcriptional regulation. Recent functional genomics and computational studies have yielded extensive datasets cataloging transcription factor binding sites (TFBS) and chromatin features, such as nucleosome positions, throughout the yeast genome. However, much of this data can be difficult to navigate or analyze efficiently. This chapter describes practical methods for the visualization, data mining, and statistical analysis of yeast promoter elements and chromatin features using two Web-accessible bioinformatics databases: ChromatinDB and Ceres. PMID:22113279

  20. Nuclear morphometry and chromatin textural characteristics of basal cell carcinoma*

    PubMed Central

    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. PMID:26734870

  1. Probing Chromatin-modifying Enzymes with Chemical Tools.

    PubMed

    Fischle, Wolfgang; Schwarzer, Dirk

    2016-03-18

    Chromatin is the universal template of genetic information in all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so-called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites of chromatin modifying enzymes and reader proteins. PMID:26845102

  2. 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

    2016-01-01

    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

  3. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  4. 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. PMID:22825423

  5. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development.

    PubMed

    Lu, Falong; Liu, Yuting; Inoue, Azusa; Suzuki, Tsukasa; Zhao, Keji; Zhang, Yi

    2016-06-01

    How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos. PMID:27259149

  6. Effect of irradiation and endogenous nucleases on rat liver chromatin

    SciTech Connect

    Gelderblom, D.; Smit, B.J.; Boehm, L.

    1984-08-01

    The assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease digests of unirradiated control nuclei. These results suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease.

  7. The chromatin landscape of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Toth, Zsolt; Brulois, Kevin; Jung, Jae U

    2013-05-01

    Kaposi's sarcoma-associated herpesvirus is an oncogenic γ-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis. PMID:23698402

  8. Asymmetric condensed dark matter

    NASA Astrophysics Data System (ADS)

    Aguirre, Anthony; Diez-Tejedor, Alberto

    2016-04-01

    We explore the viability of a boson dark matter candidate with an asymmetry between the number densities of particles and antiparticles. A simple thermal field theory analysis confirms that, under certain general conditions, this component would develop a Bose-Einstein condensate in the early universe that, for appropriate model parameters, could survive the ensuing cosmological evolution until now. The condensation of a dark matter component in equilibrium with the thermal plasma is a relativistic process, hence the amount of matter dictated by the charge asymmetry is complemented by a hot relic density frozen out at the time of decoupling. Contrary to the case of ordinary WIMPs, dark matter particles in a condensate must be lighter than a few tens of eV so that the density from thermal relics is not too large. Big-Bang nucleosynthesis constrains the temperature of decoupling to the scale of the QCD phase transition or above. This requires large dark matter-to-photon ratios and very weak interactions with standard model particles.

  9. Unsupervised pattern discovery in human chromatin structure through genomic segmentation.

    PubMed

    Hoffman, Michael M; Buske, Orion J; Wang, Jie; Weng, Zhiping; Bilmes, Jeff A; Noble, William Stafford

    2012-05-01

    We trained Segway, a dynamic Bayesian network method, simultaneously on chromatin data from multiple experiments, including positions of histone modifications, transcription-factor binding and open chromatin, all derived from a human chronic myeloid leukemia cell line. In an unsupervised fashion, we identified patterns associated with transcription start sites, gene ends, enhancers, transcriptional regulator CTCF-binding regions and repressed regions. Software and genome browser tracks are at http://noble.gs.washington.edu/proj/segway/. PMID:22426492

  10. Neutrophil extracellular traps: Is immunity the second function of chromatin?

    PubMed Central

    2012-01-01

    Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed “NETosis.” Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation. PMID:22945932

  11. 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

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

    PubMed

    Zeng, Zixian; Jiang, Jiming

    2016-06-01

    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. PMID:27142171

  13. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

  14. CCSI: a database providing chromatin–chromatin spatial interaction information

    PubMed Central

    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 PMID:26868054

  15. Modeling co-occupancy of transcription factors using chromatin features

    PubMed Central

    Liu, Liang; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

    Regulation of gene expression requires both transcription factor (TFs) and epigenetic modifications, and interplays between the two types of factors have been discovered. However study of relationships between chromatin features and TF–TF co-occupancy remains limited. Here, we revealed the relationship by first illustrating distinct profile patterns of chromatin features related to different binding events, including single TF binding and TF–TF co-occupancy of 71 TFs from five human cell lines. We further implemented statistical analyses to demonstrate the relationship by accurately predicting co-occupancy genome-widely using chromatin features including DNase I hypersensitivity, 11 histone modifications (HMs) and GC content. Remarkably, our results showed that the combination of chromatin features enables accurate predictions across the five cells. For individual chromatin features, DNase I enables high and consistent predictions. H3K27ac, H3K4me 2, H3K4me3 and H3K9ac are more reliable predictors than other HMs. Although the combination of 11 HMs achieves accurate predictions, their predictive ability varies considerably when a model obtained from one cell is applied to others, indicating relationship between HMs and TF–TF co-occupancy is cell type dependent. GC content is not a reliable predictor, but the addition of GC content to any other features enhances their predictive ability. Together, our results elucidate a strong relationship between TF–TF co-occupancy and chromatin features. PMID:26590261

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

    PubMed

    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

  17. Linker histone variants control chromatin dynamics during early embryogenesis

    PubMed Central

    Saeki, Hideaki; Ohsumi, Keita; Aihara, Hitoshi; Ito, Takashi; Hirose, Susumu; Ura, Kiyoe; Kaneda, Yasufumi

    2005-01-01

    Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency. PMID:15821029

  18. 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

  19. Can chromatin conformation technologies bring light into human molecular pathology?

    PubMed

    Kubiak, Marta; Lewandowska, Marzena Anna

    2015-01-01

    Regulation of gene expression in eukaryotes involves many complex processes, in which chromatin structure plays an important role. In addition to the epigenetic effects, such as DNA methylation and phosphorylation or histone modifications, gene expression is also controlled by the spatial organization of chromatin. For example, distant regulatory elements (enhancers, insulators) may come into direct physical interaction with target genes or other regulatory elements located in genomic regions of up to several hundred kilobases in size. Such long-range interactions result in the formation of chromatin loops. In the last several years, there has been a rapid increase in our knowledge of the spatial organization of chromatin in the nucleus through the chromosome conformation capture (3C) technology. Here we review and compare the original 3C and 3C-based methods including chromosome conformation capture-on-chip (4C), chromosome conformation capture carbon copy (5C), hi-resolution chromosome confomation capture (HiC). In this article, we discuss different aspects of how the nuclear organization of chromatin is associated with gene expression regulation and how this knowledge is useful in translational medicine and clinical applications. We demonstrate that the knowledge of the chromatin 3D organization may help understand the mechanisms of gene expression regulation of genes involved in the development of human diseases, such as CFTR (responsible for cystic fibrosis) or IGFBP3 (associated with breast cancer pathogenesis). Additionally, 3C-derivative methods have been also useful in the diagnosis of some leukemia subtypes. PMID:26328275

  20. Chromatin higher-order structure: two-start double superhelix formed by zig-zag shaped nucleosome chain with folded linker DNA.

    PubMed

    Osipova, T N; Karpova, E V; Vorob'ev, V I

    1990-08-01

    Hydrodynamic properties of chromatins differing in linker DNA length and in transcriptional activity have been studied by the method of sedimentation velocity. Oligonucleosomes of different chain length were isolated from chromatins of pigeon brain cortical neurones, rat thymus and sea urchin sperm characterized by nucleosome DNA repeat length of 165, 198 and 248 base pairs respectively. The hydrodynamic behaviour of oligonucleosomes in the dependence on the number of nucleosomes in the chain and on the ionic strength has been analysed on the basis of cylinder model. The data obtained allows one to calculate the main structural parameters of the oligonucleosomal chain: its mass per unit length, the hydrodynamic diameter of the chain, the length of the chain per nucleosome and DNA packing ratio. It is shown that hydrodynamic behaviour of nucleosome oligomers from all types of chromatins investigated at low ionic strength can be well described by the model of three-dimensional zig-zag chain with similar diameter and length of the chain per nucleosome, DNA packing ratio growing with the increase of linker DNA length. It can be achieved by unfolding the short linker DNA in neurone chromatin and by coiling the long linker DNA of sea urchin sperm chromatin into a loop. With the increase of ionic strength zig-zag shaped nucleosomal chain is condensed into a two-start double superhelix with closely arranged nucleosomes and linker DNA loops packed inside the superhelix. The suggested model is in good agreement with available experimental data and overcomes a number of difficulties which arise for the solenoid model and other models of the 30-nm chromatin fibril. PMID:2275789

  1. Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.

    PubMed

    Fu, Hongxia; Freedman, Benjamin S; Lim, Chwee Teck; Heald, Rebecca; Yan, Jie

    2011-06-01

    Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures. PMID:21369955

  2. The effect of heracleum persicum (Golpar) oil and alcoholic extracts on sperm parameters and chromatin quality in mice

    PubMed Central

    Taghizabet, Neda; Mangoli, Esmat; Anbari, Fatemeh; Masoodi, Seyed Ali; Talebi, Ali Reza; Mazrooei, Malihe

    2016-01-01

    Background: Evaluating the significance and the effects of plant-derived drugs on laboratory animal’s fertility was recognized. There was antioxidant activity reported from Heracleum persicum (Golpar). Objective: Current study aims to study the antioxidant effect of Golpar extracts on sperm parameters and chromatin quality in mice. Materials and Methods: Eighteen adult male mice were divided to 3 groups (10 wk old, 35 gr weight): group1 received hydro alcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 mg/kg, ip) and group 3 serving as the sham control group that received sterile water. Finally, left cauda epididymis of each animal was dissected and sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3) and acridine orange (AO) staining. Results: Progressive and non-progressive sperm motility were significantly increased in group 1 in comparison with group 3 (p=0.032). There was an increasing trend in progressive sperm motility and decreasing trend in non-progressive sperm motility in group 2 in comparison with group 3, but the differences were not significant (p=0.221 and p=0.144, respectively). According to the sperm chromatin quality, the results of TB and AO tests revealed significant differences (p=0.004, p=0.000, respectively) between those groups and showed that the extracts of Golpar cause DNA damage, but no differences can be observed between them in AB and CMA3 staining (p>0.05). Conclusion: The results showed that Heracleum persicum extracts may improve sperm motility. Also, it has harmful effects on sperm chromatin condensation and DNA integrity in mice. PMID:27525319

  3. 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.

  4. Localization of collisionally inhomogeneous condensates in a bichromatic optical lattice

    NASA Astrophysics Data System (ADS)

    Cheng, Yongshan; Adhikari, S. K.

    2011-02-01

    By direct numerical simulation and variational solution of the Gross-Pitaevskii equation, we studied the stationary and dynamic characteristics of a cigar-shaped, localized, collisionally inhomogeneous Bose-Einstein condensate trapped in a one-dimensional bichromatic quasiperiodic optical-lattice potential, as used in a recent experiment on the localization of a Bose-Einstein condensate [Roati , Nature (London)NATUAS0028-083610.1038/nature07071 453, 895 (2008)]. The effective potential characterizing the spatially modulated nonlinearity is obtained. It is found that the collisional inhomogeneity has influence not only on the central region but also on the tail of the Bose-Einstein condensate. The influence depends on the sign and value of the spatially modulated nonlinearity coefficient. We also demonstrate the stability of the stationary localized state by performing a standard linear stability analysis. Where possible, the numerical results are shown to be in good agreement with the variational results.

  5. Guide to a condensed form of NASTRAN

    NASA Technical Reports Server (NTRS)

    Rogers, J. L., Jr.

    1978-01-01

    A limited capability form of NASTRAN level 16 is presented to meet the needs of universities and small consulting firms. The input cards, the programming language of the direct matrix abstraction program, the plotting, the problem definition, and the modules' diagnostic messages are described. Sample problems relating to the analysis of linear static, vibration, and buckling are included. This guide can serve as a handbook for instructional courses in the use of NASTRAN or for users who need only the capability provided by the condensed form.

  6. Effect of spontaneous condensation on condensation heat transfer in the presence of non-condensable gases

    SciTech Connect

    Karl, J.; Hein, D.

    1999-07-01

    The presence of non condensable gases like nitrogen or air reduces the condensation heat transfer during condensation of binary steam mixtures. The non condensable gas accumulates in the vapor phase boundary layer and causes a high heat transfer resistance. Especially with high pressures and low water temperatures spontaneous condensation reduces heat transfer additionally. Fog forms within the steam-nitrogen boundary layer and the steam condenses on the water droplets of the fog layer. The convective mass transfer to the cooling water interface diminishes. Raman spectroscopy and film theory are used to quantify this effect locally. The calculation of overall condensation rates in large steam nitrogen systems requires to use three dimensional CFD codes. The paper presents equations to predict fog formation in the boundary layer which can be implemented in CFD codes.

  7. Chromatin-dependent repression of the Arabidopsis floral integrator genes involves plant specific PHD-containing proteins.

    PubMed

    López-González, Leticia; Mouriz, Alfonso; Narro-Diego, Laura; Bustos, Regla; Martínez-Zapater, José Miguel; Jarillo, Jose A; Piñeiro, Manuel

    2014-10-01

    The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants. PMID:25281686

  8. Condensed Matter Nuclear Science

    NASA Astrophysics Data System (ADS)

    Biberian, Jean-Paul

    2006-02-01

    1. General. A tribute to gene Mallove - the "Genie" reactor / K. Wallace and R. Stringham. An update of LENR for ICCF-11 (short course, 10/31/04) / E. Storms. New physical effects in metal deuterides / P. L. Hagelstein ... [et al.]. Reproducibility, controllability, and optimization of LENR experiments / D. J. Nagel -- 2. Experiments. Electrochemistry. Evidence of electromagnetic radiation from Ni-H systems / S. Focardi ... [et al.]. Superwave reality / I. Dardik. Excess heat in electrolysis experiments at energetics technologies / I. Dardik ... [et al.]. "Excess heat" during electrolysis in platinum/K[symbol]CO[symbol]/nickel light water system / J. Tian ... [et al.]. Innovative procedure for the, in situ, measurement of the resistive thermal coefficient of H(D)/Pd during electrolysis; cross-comparison of new elements detected in the Th-Hg-Pd-D(H) electrolytic cells / F. Celani ... [et al.]. Emergence of a high-temperature superconductivity in hydrogen cycled Pd compounds as an evidence for superstoihiometric H/D sites / A. Lipson ... [et al.]. Plasma electrolysis. Calorimetry of energy-efficient glow discharge - apparatus design and calibration / T. B. Benson and T. O. Passell. Generation of heat and products during plasma electrolysis / T. Mizuno ... [et al.]. Glow discharge. Excess heat production in Pd/D during periodic pulse discharge current in various conditions / A. B. Karabut. Beam experiments. Accelerator experiments and theoretical models for the electron screening effect in metallic environments / A. Huke, K. Czerski, and P. Heide. Evidence for a target-material dependence of the neutron-proton branching ratio in d+d reactions for deuteron energies below 20keV / A. Huke ... [et al.]. Experiments on condensed matter nuclear events in Kobe University / T. Minari ... [et al.]. Electron screening constraints for the cold fusion / K. Czerski, P. Heide, and A. Huke. Cavitation. Low mass 1.6 MHz sonofusion reactor / R. Stringham. Particle detection. Research

  9. Gravitational Condensate Stars

    NASA Astrophysics Data System (ADS)

    Mazur, P.; Mottola, E.

    The issue of the final state of the gravitational collapse will be addressed. Ishall present physical arguments to the effect that the remnant of the gravitationalcollapse of super-massive stars is a cold and dark super-dense object which isthermodynamically and dynamically stable: a Gravitational Condensate Star orQuasi Black Hole (QBH). A QBH is characterized by a huge, but not an infinite,surface redshift. This surface redshift depends universally on the total mass of aQBH and the proper thickness of a thin shell of an exotic matter described bythe Zel'dovich equation of state p = c2 . The velocity of sound in a thin shell isequal to the velocity of light. Hence, this thin shell replaces the event horizon of amathematical black hole ( = 0). Inside a thin shell the zero entropy gravitationalcondensate characterized by the cosmological equation of state p = -c2 resides.A QBH is described by a new static and spherically symmetric solution of Ein-stein's equations supplemented with the proper boundary conditions based on mi-crophysics considerations. The new solution has no singularities and no eventhorizons. Its entropy is maximized under small fluctuations and is given by thestandard hydrodynamic entropy of the thin shell which is proportional to the to-tal mass instead of the Bekenstein-Hawking entropy which is proportional to thesquare of the total mass. This resolves the paradox of an excessively high en-tropy of black holes as compared to their progenitors. The formation of such acold gravitational condensate stellar remnant very likely would require a violentcollapse process with an explosive output of energy. Some observational conse-quences of the formation of gravitational condensate stars will be described.

  10. Expansion in condensates

    SciTech Connect

    Chakrabarti, J.; Sajjad Zahir, M.

    1985-03-01

    We show that the product of local current operators in quantum chromodynamics (QCD), when expanded in terms of condensates, such as psi-barpsi, G/sup a//sub munu/ G/sup a//sub munu/, psi-barGAMMA psipsi-barGAMMApsi, f/sub a/bcG/sup a//sub munu/G/sup b//sub nualpha/ x G/sup c//sub alphamu/, etc., yields a series in Planck's constant. This, however, provides no hint that the higher terms in such an expansion may be less significant.

  11. Confinement Contains Condensates

    SciTech Connect

    Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.

    2012-03-12

    Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

  12. Condensed Plasmas under Microgravity

    NASA Technical Reports Server (NTRS)

    Morfill, G. E.; Thomas, H. M.; Konopka, U.; Rothermel, H.; Zuzic, M.; Ivlev, A.; Goree, J.; Rogers, Rick (Technical Monitor)

    1999-01-01

    Experiments under microgravity conditions were carried out to study 'condensed' (liquid and crystalline) states of a colloidal plasma (ions, electrons, and charged microspheres). Systems with approximately 10(exp 6) microspheres were produced. The observed systems represent new forms of matter--quasineutral, self-organized plasmas--the properties of which are largely unexplored. In contrast to laboratory measurements, the systems under microgravity are clearly three dimensional (as expected); they exhibit stable vortex flows, sometimes adjacent to crystalline regions, and a central 'void,' free of microspheres.

  13. The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells.

    PubMed

    Khateb, Mamduh; Fourier, Nitsan; Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

    2016-01-01

    Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron's properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

  14. The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

    PubMed Central

    Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

    2016-01-01

    Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

  15. Characterization of Nucleosome Unwrapping within Chromatin Fibers Using Magnetic Tweezers

    PubMed Central

    Chien, Fan-Tso; van der Heijden, Thijn

    2014-01-01

    Nucleosomal arrays fold into chromatin fibers and the higher-order folding of chromatin plays a strong regulatory role in all processes involving DNA access, such as transcription and replication. A fundamental understanding of such regulation requires insight into the folding properties of the chromatin fiber in molecular detail. Despite this, the structure and the mechanics of chromatin fibers remain highly disputed. Single-molecule force spectroscopy experiments have the potential to provide such insight, but interpretation of the data has been hampered by the large variations in experimental force-extension traces. Here we explore the possibility that chromatin fibers are composed of both single-turn and fully wrapped histone octamers. By characterizing the force-dependent behavior of in vitro reconstituted chromatin fibers and reanalyzing existing data, we show the unwrapping of the outer turn of nucleosomal DNA at 3 pN. We present a model composed of two freely-jointed chains, which reveals that nucleosomes within the chromatin fiber show identical force-extension behavior to mononucleosomes, indicating that nucleosome-nucleosome interactions are orders-of-magnitude smaller than previously reported and therefore can be overcome by thermal fluctuations. We demonstrate that lowering the salt concentration externally increases the wrapping energy significantly, indicative of the electrostatic interaction between the wrapped DNA and the histone octamer surface. We propose that the weak interaction between nucleosomes could allow easy access to nucleosomal DNA, while DNA unwrapping from the histone core could provide a stable yet dynamic structure during DNA maintenance. PMID:25028879

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

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    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

  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. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    PubMed

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional

  19. HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation

    PubMed Central

    2010-01-01

    Background High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin

  20. Rejoining and misrejoining of radiation-induced chromatin breaks. IV. Charged particles

    NASA Technical Reports Server (NTRS)

    Durante, M.; Furusawa, Y.; George, K.; Gialanella, G.; Greco, O.; Grossi, G.; Matsufuji, N.; Pugliese, M.; Yang, T. C.

    1998-01-01

    We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

  1. Methods to Identify Chromatin-Bound Protein Complexes: From Genome-Wide to Locus-Specific Approaches.

    PubMed

    Massie, Charles E

    2016-01-01

    High-throughput sequencing approaches coupled with functional genomics experiments have facilitated a rapid growth in our understanding of chromatin biology, from genome-wide maps of transcription factor binding and histone modifications to insights into higher order chromatin organization under specific cellular conditions. However in most cases these methods require a prior knowledge of the system of interest (e.g., targets for immunoprecipitation or modulation) and therefore are limited in their utility to identify novel components of pathways or for the study of uncharacterized pathways. Several orthologous proteomics approaches have been developed recently that bridge this gap, allowing the identification of protein complexes globally or at specific genomic loci. In this chapter the relative advantages of each approach will be explored and a detailed protocol given for DNA pull-down of a specific androgen receptor (AR) genomic target. PMID:27246338

  2. Gravitational vacuum condensate stars.

    PubMed

    Mazur, Pawel O; Mottola, Emil

    2004-06-29

    A new final state of gravitational collapse is proposed. By extending the concept of Bose-Einstein condensation to gravitational systems, a cold, dark, compact object with an interior de Sitter condensate p(v) = -rho(v) and an exterior Schwarzschild geometry of arbitrary total mass M is constructed. These regions are separated by a shell with a small but finite proper thickness l of fluid with equation of state p = +rho, replacing both the Schwarzschild and de Sitter classical horizons. The new solution has no singularities, no event horizons, and a global time. Its entropy is maximized under small fluctuations and is given by the standard hydrodynamic entropy of the thin shell, which is of the order k(B)lMc/Planck's over 2 pi, instead of the Bekenstein-Hawking entropy formula, S(BH) = 4 pi k(B)GM(2)/Planck's over 2 pi c. Hence, unlike black holes, the new solution is thermodynamically stable and has no information paradox. PMID:15210982

  3. Cosmic curvature and condensation

    NASA Technical Reports Server (NTRS)

    Harwit, Martin

    1992-01-01

    It is shown that the universe may consist of a patchwork of domains with different Riemann curvature constants k = 0, +/-1. Features of a phase transition in which flat space breaks up in a transition 2k0 - k(-) + k(+) with initial scale factors R(-) = R(+) are postulated and explored. It is shown that such a transition is energetically permitted, has the equivalent of a Curie temperature, and can lead in a natural way to the formation of voids and galaxies. It is predicted that, if the ambient universe on average is well fitted by a purely k(-) space, with only occasional domains of k(+) containing galaxies, a density parameter of (A(z sub c + 1)) super -1 should be expected, where z sub c represents the redshift of the earliest objects to have condensed, and A takes on values ranging from about 5 to 3. Present observations of quasars would suggest a density of about 0.03 or 0.05, respectively, but it could be lower if earlier condensation took place.

  4. Pion condensation in holographic QCD

    SciTech Connect

    Albrecht, Dylan; Erlich, Joshua

    2010-11-01

    We study pion condensation at zero temperature in a hard-wall holographic model of hadrons with isospin chemical potential. We find that the transition from the hadronic phase to the pion condensate phase is first order except in a certain limit of model parameters. Our analysis suggests that immediately across the phase boundary the condensate acts as a stiff medium approaching the Zel'dovich limit of equal energy density and pressure.

  5. lncRNAs: Linking RNA to Chromatin

    PubMed Central

    Rinn, John L.

    2014-01-01

    Numerous studies over the past decade have identified increasing numbers of long noncoding RNAs (lncRNAs) across many organisms. Research since has shown that lncRNAs constitute an important layer of genome regulation in diverse biological processes and disease. Here, we discuss the common emerging theme of lncRNAs interfacing with epigenetic machinery. This, in turn, modulates the activity and localization of the epigenetic machinery during cell fate specification. PMID:25085913

  6. The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis[W

    PubMed Central

    Jégu, Teddy; Latrasse, David; Delarue, Marianne; Hirt, Heribert; Domenichini, Séverine; Ariel, Federico; Crespi, Martin; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2014-01-01

    SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus. PMID:24510722

  7. 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

  8. 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. PMID:25602519

  9. Exploring chromatin organization mechanisms through its dynamic properties.

    PubMed

    Bronshtein, Irena; Kanter, Itamar; Kepten, Eldad; Lindner, Moshe; Berezin, Shirly; Shav-Tal, Yaron; Garini, Yuval

    2016-03-01

    The organization of the genome in the nucleus is believed to be crucial for different cellular functions. It is known that chromosomes fold into distinct territories, but little is known about the mechanisms that maintain these territories. To explore these mechanisms, we used various live-cell imaging methods, including single particle tracking to characterize the diffusion properties of different genomic regions in live cells. Chromatin diffusion is found to be slow and anomalous; in vast contrast, depletion of lamin A protein significantly increases chromatin motion, and the diffusion pattern of chromatin transforms from slow anomalous to fast normal. More than this, depletion of lamin A protein also affects the dynamics of nuclear bodies. Our findings indicate that chromatin motion is mediated by lamin A and we suggest that constrained chromatin mobility allows to maintain chromosome territories. Thus, the discovery of this function of nucleoplasmic lamin A proteins sheds light on the maintenance mechanism of chromosome territories in the interphase nucleus, which ensures the proper function of the genome. PMID:26854963

  10. Looking at plant cell cycle from the chromatin window

    PubMed Central

    Desvoyes, Bénédicte; Fernández-Marcos, María; Sequeira-Mendes, Joana; Otero, Sofía; Vergara, Zaida; Gutierrez, Crisanto

    2014-01-01

    The cell cycle is defined by a series of complex events, finely coordinated through hormonal, developmental and environmental signals, which occur in a unidirectional manner and end up in producing two daughter cells. Accumulating evidence reveals that chromatin is not a static entity throughout the cell cycle. In fact, there are many changes that include nucleosome remodeling, histone modifications, deposition and exchange, among others. Interestingly, it is possible to correlate the occurrence of several of these chromatin-related events with specific processes necessary for cell cycle progression, e.g., licensing of DNA replication origins, the E2F-dependent transcriptional wave in G1, the activation of replication origins in S-phase, the G2-specific transcription of genes required for mitosis or the chromatin packaging occurring in mitosis. Therefore, an emerging view is that chromatin dynamics must be considered as an intrinsic part of cell cycle regulation. In this article, we review the main features of several key chromatin events that occur at defined times throughout the cell cycle and discuss whether they are actually controlling the transit through specific cell cycle stages. PMID:25120553

  11. Histone modifications and chromatin dynamics: a focus on filamentous fungi

    PubMed Central

    Brosch, Gerald; Loidl, Peter; Graessle, Stefan

    2008-01-01

    The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. PMID:18221488

  12. Rules of Engagement for Base Excision Repair in Chromatin

    PubMed Central

    Odell, Ian D.; Wallace, Susan S.; Pederson, David S.

    2012-01-01

    Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed (HDR) and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an ‘access-repair-restore’ paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. PMID:22718094

  13. Chromatin: a tunable spring at work inside chromosomes.

    PubMed

    Ben-Haïm, E; Lesne, A; Victor, J M

    2001-11-01

    This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give a complete set of elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant) of the so-called 30-nm chromatin fiber, in terms of DNA elastic properties and geometric properties of the fiber assembly. The computation naturally embeds the fiber within a current analytical model known as the "extensible wormlike rope," allowing a straightforward prediction of the force-extension curves. We show that these elastic constants are strongly sensitive to the linker length, up to 1 bp, or equivalently to its twist, and might locally reach very low values, yielding a highly flexible and extensible domain in the fiber. In particular, the twist-stretch coupling constant, reflecting the chirality of the chromatin fiber, exhibits steep variations, and sign changes when the linker length is varied. We argue that this tunable elasticity might be a key feature for chromatin function, for instance, in the initiation and regulation of transcription. PMID:11735982

  14. 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

  15. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  16. DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

    PubMed Central

    Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael; Tsukiyama, Toshio

    2013-01-01

    Summary ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes. PMID:23478442

  17. The molecular topography of silenced chromatin in Saccharomyces cerevisiae

    PubMed Central

    Thurtle, Deborah M.; Rine, Jasper

    2014-01-01

    Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms. PMID:24493645

  18. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  19. A simple biophysical model emulates budding yeast chromosome condensation.

    PubMed

    Cheng, Tammy M K; Heeger, Sebastian; Chaleil, Raphaël A G; Matthews, Nik; Stewart, Aengus; Wright, Jon; Lim, Carmay; Bates, Paul A; Uhlmann, Frank

    2015-01-01

    Mitotic chromosomes were one of the first cell biological structures to be described, yet their molecular architecture remains poorly understood. We have devised a simple biophysical model of a 300 kb-long nucleosome chain, the size of a budding yeast chromosome, constrained by interactions between binding sites of the chromosomal condensin complex, a key component of interphase and mitotic chromosomes. Comparisons of computational and experimental (4C) interaction maps, and other biophysical features, allow us to predict a mode of condensin action. Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation. Higher order interactions between condensin binding sites explain the data less well. Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like. PMID:25922992

  20. Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

    PubMed

    Denny, Sarah K; Yang, Dian; Chuang, Chen-Hua; Brady, Jennifer J; Lim, Jing Shan; Grüner, Barbara M; Chiou, Shin-Heng; Schep, Alicia N; Baral, Jessika; Hamard, Cécile; Antoine, Martine; Wislez, Marie; Kong, Christina S; Connolly, Andrew J; Park, Kwon-Sik; Sage, Julien; Greenleaf, William J; Winslow, Monte M

    2016-07-14

    Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression. PMID:27374332