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Sample records for rapidly alters chromatin

  1. Rapid and reversible epigenome editing by endogenous chromatin regulators.

    Science.gov (United States)

    Braun, Simon M G; Kirkland, Jacob G; Chory, Emma J; Husmann, Dylan; Calarco, Joseph P; Crabtree, Gerald R

    2017-09-15

    Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

  2. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

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    Finan, John D.; Leddy, Holly A. [Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC (United States); Department of Biomedical Engineering, Duke University, Durham, NC (United States); Guilak, Farshid, E-mail: guilak@duke.edu [Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC (United States); Department of Biomedical Engineering, Duke University, Durham, NC (United States)

    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.

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

    International Nuclear Information System (INIS)

    Smith, P.J.

    1986-01-01

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

  4. The alteration of chromatin domains during damage repair induced by ionizing radiation

    International Nuclear Information System (INIS)

    Cress, A.E.; Olson, K.M.; Olson, G.B.

    1995-01-01

    Several groups previously have reported the ability of chromatin structure to influence the production of damage induced by ionizing radiation. The authors' interest has been to determine whether chromatin structural alterations exist after ionizing radiation during a repair interval. The earlier work investigated this question using biochemical techniques. The crosslinking of nuclear structural proteins to DNA after ionizing radiation was observed. In addition, they found that the chromatin structure in vitro as measured by sucrose density gradient sedimentation, was altered after ionizing radiation. These observations added to earlier studies in which digital imaging techniques showed an alteration in feulgen-positive DNA after irradiation prompted the present study. The object of this study was to detect whether the higher order structure of DNA into chromatin domains within interphase human cells was altered in interphase cells in response to a radiation induced damage. The present study takes advantage of the advances in the detection of chromatin domains in situ using DNA specific dyes and digital image processing of established human T and B cell lines

  5. Increased polyamines alter chromatin and stabilize autoantigens in autoimmune diseases

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    Wesley H. Brooks

    2013-04-01

    Full Text Available Polyamines are small cations with unique combinations of charge and length that give them many putative interactions in cells. Polyamines are essential since they are involved in replication, transcription, translation, and stabilization of macro-molecular complexes. However, polyamine synthesis competes with cellular methylation for S-adenosylmethionine, the methyl donor. Also, polyamine degradation can generate reactive molecules like acrolein. Therefore, polyamine levels are tightly controlled. This control may be compromised in autoimmune diseases since elevated polyamine levels are seen in autoimmune diseases. Here a hypothesis is presented explaining how polyamines can stabilize autoantigens. In addition, the hypothesis explains how polyamines can inappropriately activate enzymes involved in NETosis, a process in which chromatin is modified and extruded from cells as extracellular traps that bind pathogens during an immune response. This polyamine-induced enzymatic activity can lead to an increase in NETosis resulting in release of autoantigenic material and tissue damage.

  6. Controlled cooling versus rapid freezing of teratozoospermic semen samples: Impact on sperm chromatin integrity

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    Shivananda N Kalludi

    2011-01-01

    Full Text Available Aim: The present study evaluates the impact of controlled slow cooling and rapid freezing techniques on the sperm chromatin integrity in teratozoospermic and normozoospermic samples. Setting: The study was done in a university infertility clinic, which is a tertiary healthcare center serving the general population. Design: It was a prospective study designed in vitro. Materials and Methods: Semen samples from normozoospermic (N=16 and teratozoospermic (N=13 infertile men were cryopreserved using controlled cooling and rapid freezing techniques. The sperm chromatin integrity was analyzed in fresh and frozen-thawed samples. Statistical Analysis Used: Data were reported as mean and standard error (mean ± SEM of mean. The difference between two techniques was determined by a paired t-test. Results: The freeze-thaw induced chromatin denaturation was significantly (P<0.01 elevated in the post-thaw samples of normozoospermic and teratozoospermic groups. Compared to rapid freezing, there was no difference in the number of red sperms (with DNA damage by the controlled slow cooling method in both normozoospermic and teratozoospermic groups. Freeze-thaw induced sperm chromatin denaturation in teratozoospermic samples did not vary between controlled slow cooling and rapid freezing techniques. Conclusions: Since the controlled slow cooling technique involves the use of expensive instrument and is a time consuming protocol, rapid freezing can be a good alternative technique for teratozoospermic and normozoospermic samples when sperm DNA damage is a concern.

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

    International Nuclear Information System (INIS)

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B.

    2005-01-01

    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 3 (CMA 3 ). Increases in DFI (15%), DFI% (4.5-fold), and CMA 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 3 provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin

  8. Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms

    International Nuclear Information System (INIS)

    Johnson, E.M.; Sterner, R.; Allfrey, V.G.

    1987-01-01

    Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo[2-tritium]acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription

  9. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

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    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

  10. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

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    Gábor Vasas

    2013-09-01

    Full Text Available 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.

  11. Exposure to Hycanthone alters chromatin structure around specific gene functions and specific repeats in Schistosoma mansoni

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    David eRoquis

    2014-07-01

    Full Text Available Schistosoma mansoni is a parasitic plathyhelminth responsible for intestinal schistosomiasis (or bilharziasis, a disease affecting 67 million people worldwide and causing an important economic burden. The schistosomicides hycanthone, and its later proxy oxamniquine, were widely used for treatments in endemic areas during the 20th century. Recently, the mechanism of action, as well as the genetic origin of a stably and Mendelian inherited resistance for both drugs was elucidated in two strains. However, several observations suggested early on that alternative mechanisms might exist, by which resistance could be induced for these two drugs in sensitive lines of schistosomes. This induced resistance appeared rapidly, within the first generation, but was metastable (not stably inherited. Epigenetic inheritance could explain such a phenomenon and we therefore re-analyzed the historical data with our current knowledge of epigenetics. In addition, we performed new experiments such as ChIP-seq on hycanthone treated worms. We found distinct chromatin structure changes between sensitive worms and induced resistant worms from the same strain. No specific pathway was discovered, but genes in which chromatin structure modification were observed are mostly associated with transport and catabolism, which makes sense in the context of the elimination of the drug. Specific differences were observed in the repetitive compartment of the genome. We finally describe what types of experiments are needed to understand the complexity of heritability that can be based on genetic and/or epigenetic mechanisms for drug resistance in schistosomes.

  12. High-order chromatin architecture shapes the landscape of chromosomal alterations in cancer

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    Fudenberg, Geoffrey; Getz, Gad; Meyerson, Matthew; Mirny, Leonid

    2012-02-01

    The rapid growth of cancer genome structural information provides an opportunity for a better understanding of the mutational mechanisms of genomic alterations in cancer and the forces of selection that act upon them. Here we test the evidence for two major forces, spatial chromosome structure and purifying (or negative) selection, that shape the landscape of somatic copy-number alterations (SCNAs) in cancer (Beroukhim et al, 2010). Using a maximum likelihood framework we compare SCNA maps and three-dimensional genome architecture as determined by genome-wide chromosome conformation capture (HiC) and described by the proposed fractal-globule (FG) model (Lieberman-Aiden and Van Berkum et al, 2009). This analysis provides evidence that the distribution of chromosomal alterations in cancer is spatially related to three-dimensional genomic architecture and additionally suggests that purifying selection as well as positive selection shapes the landscape of SCNAs during somatic evolution of cancer cells.

  13. Accumulation of DNA damage-induced chromatin alterations in tissue-specific stem cells: the driving force of aging?

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    Nadine Schuler

    Full Text Available Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs, hair follicle stem cells (HFSCs in mouse epidermis were analyzed for age-related DNA damage response (DDR. We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.

  14. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

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    Máthé, Csaba; Mikóné 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 studi...

  15. Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer

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    Alfonso Urbanucci

    2017-06-01

    Full Text Available Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4 have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC. Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC. We show that the deregulation of androgen receptor (AR expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10 for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

  16. Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA.

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    Richards, W L; Song, M K; Krutzsch, H; Evarts, R P; Marsden, E; Thorgeirsson, S S

    1985-07-01

    We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.

  17. Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure.

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    Veazey, Kylee J; Wang, Haiqing; Bedi, Yudhishtar S; Skiles, William M; Chang, Richard Cheng-An; Golding, Michael C

    2017-05-01

    Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact that alcohol and other drugs of abuse exert consistently notes a disconnection between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the 'histone code' induced by prenatal exposures to alcohol implicitly subvert gene expression, or whether the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation, or affect the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest that changes

  18. Alpha radiation-induced alterations of the proliferation kinetics, chromatin structure and gene expression in mammalian cells

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    Hieber, L.

    1983-01-01

    Exponentially growing mammalian cells were exposed to 3.4 MeV alpha particles. The chromatin of cells arrested in G2 by alpha irradiation was severely damaged, though all cells were still capable to condensate their chromatin after fusion with mitotic cells. In addition to the common types of aberrations (breaks, gaps, dicentrics and exchanges) cells were found possessing one or more chromosomes with long stretches of undercondensed chromatin. Repair of these lesions was indicated by site specific unscheduled DNA synthesis and by the observation that condensation of these regions improved during G2 arrest. Furthermore, during G2 arrest the synthesis of two cellular proteins was stimulated. This was studied by two-dimensional gel electrophoresis of 35 S-methionine labeled cellular proteins. All these findings provided evidence that radiation-induced G2 arrest is caused by chromatin damage, which prevents regular chromosome condensation for mitosis. (orig./MG) [de

  19. Reprogramming chromatin

    DEFF Research Database (Denmark)

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

    attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming...... and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors...

  20. Interaction of a common painkiller piroxicam and copper-piroxicam with chromatin causes structural alterations accompanied by modulation at the epigenomic/genomic level.

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    Goswami, Sathi; Sanyal, Sulagna; Chakraborty, Payal; Das, Chandrima; Sarkar, Munna

    2017-08-01

    NSAIDs are the most common class of painkillers and anti-inflammatory agents. They also show other functions like chemoprevention and chemosuppression for which they act at the protein but not at the genome level since they are mostly anions at physiological pH, which prohibit their approach to the poly-anionic DNA. Complexing the drugs with bioactive metal obliterate their negative charge and allow them to bind to the DNA, thereby, opening the possibility of genome level interaction. To test this hypothesis, we present the interaction of a traditional NSAID, Piroxicam and its copper complex with core histone and chromatin. Spectroscopy, DLS, and SEM studies were applied to see the effect of the interaction on the structure of histone/chromatin. This was coupled with MTT assay, immunoblot analysis, confocal microscopy, micro array analysis and qRT-PCR. The interaction of Piroxicam and its copper complex with histone/chromatin results in structural alterations. Such structural alterations can have different biological manifestations, but to test our hypothesis, we have focused only on the accompanied modulations at the epigenomic/genomic level. The complex, showed alteration of key epigenetic signatures implicated in transcription in the global context, although Piroxicam caused no significant changes. We have correlated such alterations caused by the complex with the changes in global gene expression and validated the candidate gene expression alterations. Our results provide the proof of concept that DNA binding ability of the copper complexes of a traditional NSAID, opens up the possibility of modulations at the epigenomic/genomic level. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Diet rapidly and reproducibly alters the human gut microbiome

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    David, Lawrence A.; Maurice, Corinne F.; Carmody, Rachel N.; Gootenberg, David B.; Button, Julie E.; Wolfe, Benjamin E.; Ling, Alisha V.; Devlin, A. Sloan; Varma, Yug; Fischbach, Michael A.; Biddinger, Sudha B.; Dutton, Rachel J.; Turnbaugh, Peter J.

    2013-01-01

    Long-term diet influences the structure and activity of the trillions of microorganisms residing in the human gut1–5, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here, we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila, and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale, and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals2, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids, and the outgrowth of microorganisms capable of triggering inflammatory bowel disease6. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles. PMID:24336217

  2. Extinction order and altered community structure rapidly disrupt ecosystem functioning.

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    Larsen, Trond H; Williams, Neal M; Kremen, Claire

    2005-05-01

    By causing extinctions and altering community structure, anthropogenic disturbances can disrupt processes that maintain ecosystem integrity. However, the relationship between community structure and ecosystem functioning in natural systems is poorly understood. Here we show that habitat loss appeared to disrupt ecosystem functioning by affecting extinction order, species richness and abundance. We studied pollination by bees in a mosaic of agricultural and natural habitats in California and dung burial by dung beetles on recently created islands in Venezuela. We found that large-bodied bee and beetle species tended to be both most extinction-prone and most functionally efficient, contributing to rapid functional loss. Simulations confirmed that extinction order led to greater disruption of function than predicted by random species loss. Total abundance declined with richness and also appeared to contribute to loss of function. We demonstrate conceptually and empirically how the non-random response of communities to disturbance can have unexpectedly large functional consequences.

  3. Effects of fast neutrons on chromatin: dependence on chromatin structure

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    Radu, L. [Dept. of Molecular Genetics, V. Babes National Inst., Bd. Timisoara, Bucharest (Romania); Constantinescu, B. [Dept. of Cyclotron, H. Hulubei National Inst., Bucharest (Romania); Gazdaru, D. [Dept. of Biophysics, Physics Faculty, Univ. of Bucharest (Romania)

    2002-07-01

    The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin-proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)-heterochromatin (compact) ratio. (author)

  4. Effects of fast neutrons on chromatin: dependence on chromatin structure

    International Nuclear Information System (INIS)

    Radu, L.; Constantinescu, B.; Gazdaru, D.

    2002-01-01

    The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin-proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)-heterochromatin (compact) ratio. (author)

  5. Chromatin Hydrodynamics

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

    2014-01-01

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory—the two-fluid model—in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model—the Maxwell fluid—for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity. PMID:24806919

  6. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

    2017-01-01

    Inheritance of the DNA sequence and its proper organization into chromatin is fundamental for genome stability and function. Therefore, how specific chromatin structures are restored on newly synthesized DNA and transmitted through cell division remains a central question to understand cell fate...... choices and self-renewal. Propagation of genetic information and chromatin-based information in cycling cells entails genome-wide disruption and restoration of chromatin, coupled with faithful replication of DNA. In this chapter, we describe how cells duplicate the genome while maintaining its proper...... organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

  7. A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests.

    Directory of Open Access Journals (Sweden)

    Frederick A Schroeder

    Full Text Available Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary--albeit often ineffective--treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60, a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA, a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing

  8. Chromatin-modifying proteins in cancer

    DEFF Research Database (Denmark)

    Fog, Cathrine K; Jensen, Klaus T; Lund, Anders Henrik

    2007-01-01

    -despite the fact that all cells in the organism contain the same genetic information. A large amount of data gathered over the last decades has demonstrated that deregulation of chromatin-modifying proteins is etiologically involved in the development and progression of cancer. Here we discuss how epigenetic...... alterations influence cancer development and review known cancer-associated alterations in chromatin-modifying proteins....

  9. Chromatin in embryonic stem cell neuronal differentiation.

    Science.gov (United States)

    Meshorer, E

    2007-03-01

    Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

  10. Radiation response and chromatin dynamics

    International Nuclear Information System (INIS)

    Ikura, Tsuyoshi

    2009-01-01

    Described is a recent progress in studies of chromatin structural alterations induced by DNA damage by radiation. DNA in eukaryotes exists in the chromatin structure and different mechanisms of response to damage and repair of DNA from those in prokaryotes have been recognized. Chromatin is composed from its unit structure of mono-nucleosome, which is formed from DNA and an octamer of core histones of H2A, H2B, H3 and H4. When DNA is damaged, histone structural alterations are required for repair factors and checkpoint proteins to access the damaged site. At the actual genome damage, chemical modification of histone to work as a code occurs dependently on the damage where chromatin remodeling factors and histone chaperone participate for structural alteration and remodeling. As well, the exchange of histone variants and fluidization of histones are recently reported. Known chemical modification involves phosphorylation, acetylation and ubiquitination of H2AX (a variant of H2A), and acetylation and methylation of H3. Each complex of TIP60, NuA4 and INO80 is known to be included in the regulation of chromatin with damaged/repaired DNA for remodeling, but little is known about recruitment of the factors concerned at the damage site. Regulatory mechanisms in above chromatin dynamics with consideration of quality and timing of radiation should be further elucidated for understanding the precise response to DNA damage. (K.T.)

  11. Gender and rapid alterations of hemispheric dominance during planning.

    Science.gov (United States)

    Schuepbach, Daniel; Skotchko, Tatjana; Duschek, Stefan; Theodoridou, Anastasia; Grimm, Simone; Boeker, Heinz; Seifritz, Erich

    2012-01-01

    Mental planning and carrying out a plan provoke specific cerebral hemodynamic responses. Gender aspects of hemispheric laterality using rapid cerebral hemodynamics have not been reported. Here, we applied functional transcranial Doppler sonography to examine lateralization of cerebral hemodynamics of the middle cerebral arteries of 28 subjects (14 women and 14 men) performing a standard planning task. There were easy and difficult problems, and mental planning without motor activity was separated from movement execution. Difficult mental planning elicited lateralization to the right hemisphere after 2 or more seconds, a feature that was not observed during movement execution. In females, there was a dominance to the left hemisphere during movement execution. Optimized problem solving yielded an increased laterality change to the right during mental planning. Gender-related hemispheric dominance appears to be condition-dependent, and change of laterality to the right may play a role in optimized performance. Results are of relevance when considering laterality from a perspective of performance enhancement of higher cognitive functions, and also of psychiatric disorders with cognitive dysfunctions and abnormal lateralization patterns such as schizophrenia. Copyright © 2012 S. Karger AG, Basel.

  12. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations.

    Directory of Open Access Journals (Sweden)

    Lee A Hadwiger

    2015-06-01

    Full Text Available Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid nonhost resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph and the susceptibility of pea to F. solani f sp. pisi (Fspi has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to the chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction.

  13. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations

    Science.gov (United States)

    Hadwiger, Lee A.

    2015-01-01

    Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid “nonhost” resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph) and the susceptibility of pea to F. solani f sp. pisi (Fspi) has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR) genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction. PMID:26124762

  14. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations.

    Science.gov (United States)

    Hadwiger, Lee A

    2015-01-01

    Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid "nonhost" resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph) and the susceptibility of pea to F. solani f sp. pisi (Fspi) has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR) genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction.

  15. Altering plasma sodium concentration rapidly changes blood pressure during haemodialysis.

    Science.gov (United States)

    Suckling, Rebecca J; Swift, Pauline A; He, Feng J; Markandu, Nirmala D; MacGregor, Graham A

    2013-08-01

    Plasma sodium is increased following each meal containing salt. There is an increasing interest in the effects of plasma sodium concentration, and it has been suggested that it may have direct effects on blood pressure (BP) and possibly influences endothelial function. Experimental increases of plasma sodium concentration rapidly raise BP even when extracellular volume falls. Ten patients with end-stage renal failure established on haemodialysis were studied during the first 2 h of dialysis without fluid removal during this period. They were randomized to receive haemodialysis with (i) dialysate sodium concentration prescribed to 135 mmol/L and (ii) 145 mmol/L in random order in a prospective, single-blinded crossover study. BP measurements and blood samples were taken every 30 min. Pre-dialysis sitting BP was 137/76 ± 7/3 mmHg. Lower dialysate sodium concentration (135 mmol/L) reduced plasma sodium concentration [139.49 ± 0.67 to 135.94 ± 0.52 mmol/L (P area under the curve (AUC) 15823.50 ± 777.15 (mmHg)min] compared with 145 mmol/L [AUC 17018.20 ± 1102.17 (mmHg)min], mean difference 1194.70 ± 488.41 (mmHg)min, P < 0.05. There was a significant positive relationship between change in plasma sodium concentration and change in systolic BP. This direct relationship suggests that a fall of 1 mmol/L in plasma sodium concentration would be associated with a 1.7 mmHg reduction in systolic BP (P < 0.05). The potential mechanism for the increase in BP seen with salt intake may be through small but significant changes in plasma sodium concentration.

  16. UV-induced structural changes in chromatin

    International Nuclear Information System (INIS)

    Lang, H.; Zimmer, C.; Vengerov, Yu.Yu.

    1985-01-01

    UV-induced structural alterations of chromatin were studied by means of CD, electron microscopic, and gel electrophoretic measurements. The results indicate that chromatin undergoes serious structural changes after irradiation even at very low fluences. In the low fluence range the structural transitions from the higher ordered chromatin structure to the unfolded state occur without detectable changes in the content of histone H1 and of the core histones. Histone H1 disappears only at fluences above 10 kJ/m 2 . Furthermore, DNA in chromatin is much more sensitive against UV-irradiation and shows a higher degree of strand scission relative to free DNA. While fragmentation in free DNA occurs at fluences above 15 kJ/m 2 , it occurs even at 5.5 kJ/m 2 in the case of chromatin. The biological meaning of the observed UV-induced structural alterations of chromatin is discussed. (author)

  17. Chromatin remodeling, development and disease

    International Nuclear Information System (INIS)

    Ko, Myunggon; Sohn, Dong H.; Chung, Heekyoung; Seong, Rho H.

    2008-01-01

    Development is a stepwise process in which multi-potent progenitor cells undergo lineage commitment, differentiation, proliferation and maturation to produce mature cells with restricted developmental potentials. This process is directed by spatiotemporally distinct gene expression programs that allow cells to stringently orchestrate intricate transcriptional activation or silencing events. In eukaryotes, chromatin structure contributes to developmental progression as a blueprint for coordinated gene expression by actively participating in the regulation of gene expression. Changes in higher order chromatin structure or covalent modification of its components are considered to be critical events in dictating lineage-specific gene expression during development. Mammalian cells utilize multi-subunit nuclear complexes to alter chromatin structure. Histone-modifying complex catalyzes covalent modifications of histone tails including acetylation, methylation, phosphorylation and ubiquitination. ATP-dependent chromatin remodeling complex, which disrupts histone-DNA contacts and induces nucleosome mobilization, requires energy from ATP hydrolysis for its catalytic activity. Here, we discuss the diverse functions of ATP-dependent chromatin remodeling complexes during mammalian development. In particular, the roles of these complexes during embryonic and hematopoietic development are reviewed in depth. In addition, pathological conditions such as tumor development that are induced by mutation of several key subunits of the chromatin remodeling complex are discussed, together with possible mechanisms that underlie tumor suppression by the complex

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

    Science.gov (United States)

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

    2016-10-06

    Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling.

    Science.gov (United States)

    Wieczorek, Dagmar; Bögershausen, Nina; Beleggia, Filippo; Steiner-Haldenstätt, Sabine; Pohl, Esther; Li, Yun; Milz, Esther; Martin, Marcel; Thiele, Holger; Altmüller, Janine; Alanay, Yasemin; Kayserili, Hülya; Klein-Hitpass, Ludger; Böhringer, Stefan; Wollstein, Andreas; Albrecht, Beate; Boduroglu, Koray; Caliebe, Almuth; Chrzanowska, Krystyna; Cogulu, Ozgur; Cristofoli, Francesca; Czeschik, Johanna Christina; Devriendt, Koenraad; Dotti, Maria Teresa; Elcioglu, Nursel; Gener, Blanca; Goecke, Timm O; Krajewska-Walasek, Malgorzata; Guillén-Navarro, Encarnación; Hayek, Joussef; Houge, Gunnar; Kilic, Esra; Simsek-Kiper, Pelin Özlem; López-González, Vanesa; Kuechler, Alma; Lyonnet, Stanislas; Mari, Francesca; Marozza, Annabella; Mathieu Dramard, Michèle; Mikat, Barbara; Morin, Gilles; Morice-Picard, Fanny; Ozkinay, Ferda; Rauch, Anita; Renieri, Alessandra; Tinschert, Sigrid; Utine, G Eda; Vilain, Catheline; Vivarelli, Rossella; Zweier, Christiane; Nürnberg, Peter; Rahmann, Sven; Vermeesch, Joris; Lüdecke, Hermann-Josef; Zeschnigk, Michael; Wollnik, Bernd

    2013-12-20

    Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.

  20. Chromatin is wonderful stuff.

    NARCIS (Netherlands)

    van Driel, R.

    2007-01-01

    Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all

  1. Chromatin Structure and Function

    CERN Document Server

    Wolffe, Alan P

    1999-01-01

    The Third Edition of Chromatin: Structure and Function brings the reader up-to-date with the remarkable progress in chromatin research over the past three years. It has been extensively rewritten to cover new material on chromatin remodeling, histone modification, nuclear compartmentalization, DNA methylation, and transcriptional co-activators and co-repressors. The book is written in a clear and concise fashion, with 60 new illustrations. Chromatin: Structure and Function provides the reader with a concise and coherent account of the nature, structure, and assembly of chromatin and its active

  2. Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

    International Nuclear Information System (INIS)

    Pal-Bhadra, Manika; Bhadra, Utpal; Ramaiah, M Janaki; Reddy, T Lakshminarayan; Krishnan, Anita; Pushpavalli, SNCVL; Babu, K Suresh; Tiwari, Ashok K; Rao, J Madhusudana; Yadav, Jhillu S

    2012-01-01

    Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21 WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21 WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK

  3. Effect of hyperthermia on replicating chromatin

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  4. The chromatin remodeler SPLAYED regulates specific stress signaling pathways.

    Directory of Open Access Journals (Sweden)

    Justin W Walley

    2008-12-01

    Full Text Available Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD is required for the expression of selected genes downstream of the jasmonate (JA and ethylene (ET signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.

  5. Alterações de cromatina em espermatozóides de ovinos e caprinos avaliadas por azul de toluidina e alaranjado de acridina Chromatin alterations in ram and goat spermatozoa evaluated by toluidine blue and acridine orange

    Directory of Open Access Journals (Sweden)

    Cristina de Figueiredo Kamimura

    2010-02-01

    Full Text Available Reprodutores com espermograma normal podem se comportar como subférteis ou passarem por períodos de subfertilidade. As alterações na cromatina dos espermatozóides são possíveis explicações encontradas para tais comportamentos. Conduziu-se este trabalho, com o objetivo de testar a eficiência de azul de toluidina (AT e do alaranjado de acridina (AA na identificação de alterações na compactação de cromatina em espermatozóides de ovinos e caprinos, além de avaliar a correlação entre essas alterações e as de morfologia espermática. Para tal, foram avaliadas amostras de sêmen de 15 ovinos e de 15 caprinos, com dez repetições para cada método por animal. Calcularam-se a média, o desvio padrão (DP e o coeficiente de variação (CV para cada técnica e animal. Utilizou-se o teste t-Student para avaliar diferença entre as médias obtidas nos dois métodos. Também foram calculados a correlação de Pearson e os coeficientes kappa ponderado e não ponderado para avaliar a concordância entre os métodos com AT e AA. Foi verificado que nem sempre as anomalias morfológicas de cabeça são acompanhadas por alterações na cromatina identificáveis pelos métodos utilizados neste trabalho. O método AT é mais estável e possui maior sensibilidade do que AA para ambas as espécies, sendo o mais indicado para caprinos. Contudo, em razão de apresentar repetibilidade muito baixa, ambos os métodos não são indicados para avaliação espermática em ovinos.Males with normal spermogram can behave as subfertile or pass for periods of subfertility. Chromatin alterations of spermatozoa can account for such behavior. The objective of the present work was to test the efficacy of toluidine blue (TB and acridine orange (AO in the identification of alterations in chromatin compaction in spermatozoa from rams and goats, in addition to evaluate the correlation between those alterations and the ones of spermatic morphology. In order to do that

  6. Chromatin meets its organizers.

    Science.gov (United States)

    Bodnar, Megan S; Spector, David L

    2013-06-06

    Chromatin organization and gene-gene interactions are critical components of carrying out developmental programs. Phillips-Cremins et al. identify a series of unexpected architectural proteins that work in a combinatorial manner to functionally organize chromatin in a cell-type-specific manner at the submegabase-length scale. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice.

    Science.gov (United States)

    Saul, M C; Majdak, P; Perez, S; Reilly, M; Garland, T; Rhodes, J S

    2017-03-01

    Although exercise is critical for health, many lack the motivation to exercise, and it is unclear how motivation might be increased. To uncover the molecular underpinnings of increased motivation for exercise, we analyzed the transcriptome of the striatum in four mouse lines selectively bred for high voluntary wheel running and four non-selected control lines. The striatum was dissected and RNA was extracted and sequenced from four individuals of each line. We found multiple genes and gene systems with strong relationships to both selection and running history over the previous 6 days. Among these genes were Htr1b, a serotonin receptor subunit and Slc38a2, a marker for both glutamatergic and γ-aminobutyric acid (GABA)-ergic signaling. System analysis of the raw results found enrichment of transcriptional regulation and kinase genes. Further, we identified a splice variant affecting the Wnt-related Golgi signaling gene Tmed5. Using coexpression network analysis, we found a cluster of interrelated coexpression modules with relationships to running behavior. From these modules, we built a network correlated with running that predicts a mechanistic relationship between transcriptional regulation by nucleosome structure and Htr1b expression. The Library of Integrated Network-Based Cellular Signatures identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta-opioid receptor antagonist 7-benzylidenenaltrexone as potential compounds for increasing the motivation to run. Taken together, our findings support a neurobiological framework of exercise motivation where chromatin state leads to differences in dopamine signaling through modulation of both the primary neurotransmitters glutamate and GABA, and by neuromodulators such as serotonin. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  8. Heterogeneous chromatin target model

    International Nuclear Information System (INIS)

    Watanabe, Makoto

    1996-01-01

    The higher order structure of the entangled chromatin fibers in a chromosome plays a key role in molecular control mechanism involved in chromosome mutation due to ionizing radiations or chemical mutagens. The condensed superstructure of chromatin is not so rigid and regular as has been postulated in general. We have proposed a rheological explanation for the flexible network system ('chromatin network') that consists of the fluctuating assembly of nucleosome clusters linked with supertwisting DNA in a chromatin fiber ('Supertwisting Particulate Model'). We have proposed a 'Heterosensitive Target Model' for cellular radiosensitivity that is a modification of 'Heterogeneous Target Model'. The heterogeneity of chromatin target is derived from the highly condensed organization of chromatin segments consist of unstable and fragile sites in the fluctuating assembly of nucleosome clusters, namely 'supranucleosomal particles' or 'superbeads'. The models have been principally supported by our electron microscopic experiments employing 'surface - spreading whole - mount technique' since 1967. However, some deformation and artifacts in the chromatin structure are inevitable with these electron microscopic procedures. On the contrary, the 'atomic force microscope (AFM)' can be operated in liquid as well as in the air. A living specimen can be examined without any preparative procedures. Micromanipulation of the isolated chromosome is also possible by the precise positional control of a cantilever on the nanometer scale. The living human chromosomes were submerged in a solution of culture medium and observed by AFM using a liquid immersion cell. The surface - spreading whole - mount technique was applicable for this observation. The particulate chromatin segments of nucleosome clusters were clearly observed within mitotic human chromosomes in a living hydrated condition. These findings support the heterogeneity of chromatin target in a living cell. (J.P.N.)

  9. Rapid Alterations in Perirenal Adipose Tissue Transcriptomic Networks with Cessation of Voluntary Running.

    Directory of Open Access Journals (Sweden)

    Gregory N Ruegsegger

    Full Text Available In maturing rats, the growth of abdominal fat is attenuated by voluntary wheel running. After the cessation of running by wheel locking, a rapid increase in adipose tissue growth to a size that is similar to rats that have never run (i.e. catch-up growth has been previously reported by our lab. In contrast, diet-induced increases in adiposity have a slower onset with relatively delayed transcriptomic responses. The purpose of the present study was to identify molecular pathways associated with the rapid increase in adipose tissue after ending 6 wks of voluntary running at the time of puberty. Age-matched, male Wistar rats were given access to running wheels from 4 to 10 weeks of age. From the 10th to 11th week of age, one group of rats had continued wheel access, while the other group had one week of wheel locking. Perirenal adipose tissue was extracted, RNA sequencing was performed, and bioinformatics analyses were executed using Ingenuity Pathway Analysis (IPA. IPA was chosen to assist in the understanding of complex 'omics data by integrating data into networks and pathways. Wheel locked rats gained significantly more fat mass and significantly increased body fat percentage between weeks 10-11 despite having decreased food intake, as compared to rats with continued wheel access. IPA identified 646 known transcripts differentially expressed (p < 0.05 between continued wheel access and wheel locking. In wheel locked rats, IPA revealed enrichment of transcripts for the following functions: extracellular matrix, macrophage infiltration, immunity, and pro-inflammatory. These findings suggest that increases in visceral adipose tissue that accompanies the cessation of pubertal physical activity are associated with the alteration of multiple pathways, some of which may potentiate the development of pubertal obesity and obesity-associated systemic low-grade inflammation that occurs later in life.

  10. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    Directory of Open Access Journals (Sweden)

    Thiel Cora S

    2012-01-01

    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  11. Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

    OpenAIRE

    Tran, Vuong; Gan, Qiang; Chen, Xin

    2012-01-01

    Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. ...

  12. Rapid plant evolution in the presence of an introduced species alters community composition.

    Science.gov (United States)

    Smith, David Solance; Lau, Matthew K; Jacobs, Ryan; Monroy, Jenna A; Shuster, Stephen M; Whitham, Thomas G

    2015-10-01

    Because introduced species may strongly interact with native species and thus affect their fitness, it is important to examine how these interactions can cascade to have ecological and evolutionary consequences for whole communities. Here, we examine the interactions among introduced Rocky Mountain elk, Cervus canadensis nelsoni, a common native plant, Solidago velutina, and the diverse plant-associated community of arthropods. While introduced species are recognized as one of the biggest threats to native ecosystems, relatively few studies have investigated an evolutionary mechanism by which introduced species alter native communities. Here, we use a common garden design that addresses and supports two hypotheses. First, native S. velutina has rapidly evolved in the presence of introduced elk. We found that plants originating from sites with introduced elk flowered nearly 3 weeks before plants originating from sites without elk. Second, evolution of S. velutina results in a change to the plant-associated arthropod community. We found that plants originating from sites with introduced elk supported an arthropod community that had ~35 % fewer total individuals and a different species composition. Our results show that the impacts of introduced species can have both ecological and evolutionary consequences for strongly interacting species that subsequently cascade to affect a much larger community. Such evolutionary consequences are likely to be long-term and difficult to remediate.

  13. Global chromatin fibre compaction in response to DNA damage

    International Nuclear Information System (INIS)

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

    2011-01-01

    Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► 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 (γH2AX), 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 γH2AX 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 linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

  14. Dynamics of Histone Tails within Chromatin

    Science.gov (United States)

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

    2012-02-01

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

  15. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  16. Chromatin maturation depends on continued DNA-replication

    International Nuclear Information System (INIS)

    Schlaeger, E.J.; Puelm, W.; Knippers, R.

    1983-01-01

    The structure of [ 3 H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [ 3 H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. The authors conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation. (Auth.)

  17. Reprogramming the chromatin landscape

    DEFF Research Database (Denmark)

    Miranda, Tina B; Voss, Ty C; Sung, Myong-Hee

    2013-01-01

    , mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER...... and GR. Upon induction, GR modulates access of ER to specific sites in the genome by reorganization of the chromatin configuration for these elements. Binding to these newly accessible sites occurs either by direct recognition of ER response elements or indirectly through interactions with other factors...

  18. Dietary polyphenols and chromatin remodeling.

    Science.gov (United States)

    Russo, Gian Luigi; Vastolo, Viviana; Ciccarelli, Marco; Albano, Luigi; Macchia, Paolo Emidio; Ungaro, Paola

    2017-08-13

    Polyphenols are the most abundant phytochemicals in fruits, vegetables, and plant-derived beverages. Recent findings suggest that polyphenols display the ability to reverse adverse epigenetic regulation involved in pathological conditions, such as obesity, metabolic disorder, cardiovascular and neurodegenerative diseases, and various forms of cancer. Epigenetics, defined as heritable changes to the transcriptome, independent from those occurring in the genome, includes DNA methylation, histone modifications, and posttranscriptional gene regulation by noncoding RNAs. Sinergistically and cooperatively, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Such induced epigenetic changes can be inherited during cell division, resulting in permanent maintenance of the acquired phenotype, but they may also occur throughout an individual life-course and may ultimately influence phenotypic outcomes (health and disease risk). In the last decade, a number of studies have shown that nutrients can affect metabolic traits by altering the structure of chromatin and directly regulate both transcription and translational processes. In this context, dietary polyphenol-targeted epigenetics becomes an attractive approach for disease prevention and intervention. Here, we will review how polyphenols, including flavonoids, curcuminoids, and stilbenes, modulate the establishment and maintenance of key epigenetic marks, thereby influencing gene expression and, hence, disease risk and health.

  19. Proteomic interrogation of human chromatin.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

    Full Text Available Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the "Chromatome" is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Uppal, Timsy [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Jha, Hem C. [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States); Verma, Subhash C. [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Robertson, Erle S., E-mail: erle@mail.med.upenn.edu [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States)

    2015-01-14

    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.

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

    International Nuclear Information System (INIS)

    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

  2. Changes to dryland rainfall result in rapid moss mortality and altered soil fertility

    Science.gov (United States)

    Reed, Sasha C.; Coe, Kirsten K.; Sparks, Jed P.; Housman, David C.; Zelikova, Tamara J.; Belnap, Jayne

    2012-01-01

    Arid and semi-arid ecosystems cover ~40% of Earth’s terrestrial surface, but we know little about how climate change will affect these widespread landscapes. Like many drylands, the Colorado Plateau in southwestern United States is predicted to experience elevated temperatures and alterations to the timing and amount of annual precipitation. We used a factorial warming and supplemental rainfall experiment on the Colorado Plateau to show that altered precipitation resulted in pronounced mortality of the widespread moss Syntrichia caninervis. Increased frequency of 1.2 mm summer rainfall events reduced moss cover from ~25% of total surface cover to fertility. Mosses are important members in many dryland ecosystems and the community changes observed here reveal how subtle modifications to climate can affect ecosystem structure and function on unexpectedly short timescales. Moreover, mortality resulted from increased precipitation through smaller, more frequent events, underscoring the importance of precipitation event size and timing, and highlighting our inadequate understanding of relationships between climate and ecosystem function in drylands.

  3. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    International Nuclear Information System (INIS)

    Wang Liu; Zheng Aihua; Yi Ling; Xu Chongren; Ding Mingxiao; Deng Hongkui

    2004-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation

  4. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner

    DEFF Research Database (Denmark)

    Alexiadis, V; Waldmann, T; Andersen, Jens S.

    2000-01-01

    The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology...

  5. Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer

    Directory of Open Access Journals (Sweden)

    Nicholas C. Gomez

    2016-11-01

    Full Text Available Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably, we found that the chromatin environment of Ewing sarcoma, a mesenchymally derived tumor, is shared with primary mesenchymal stem cells (MSCs. Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements, a feature associated with differentiation and oncogenesis.

  6. Chromatin dynamics in genome stability

    DEFF Research Database (Denmark)

    Nair, Nidhi; Shoaib, Muhammad; Sørensen, Claus Storgaard

    2017-01-01

    Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote...... access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance...... of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage....

  7. Gene alterations at Drosophila inversion breakpoints provide prima facie evidence for natural selection as an explanation for rapid chromosomal evolution

    Science.gov (United States)

    2012-01-01

    Background Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. Results In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. Conclusions D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution. PMID:22296923

  8. Gene alterations at Drosophila inversion breakpoints provide prima facie evidence for natural selection as an explanation for rapid chromosomal evolution.

    Science.gov (United States)

    Guillén, Yolanda; Ruiz, Alfredo

    2012-02-01

    Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

  9. Gene alterations at Drosophila inversion breakpoints provide prima facie evidence for natural selection as an explanation for rapid chromosomal evolution

    Directory of Open Access Journals (Sweden)

    Guillén Yolanda

    2012-02-01

    Full Text Available Abstract Background Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. Results In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. Conclusions D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

  10. Chromatin Flavors: Chromatin composition and domain organization in Drosophila melanogaster

    NARCIS (Netherlands)

    J.G. van Bemmel (Joke)

    2012-01-01

    textabstractChromatin was originally identified by W. Flemming in 1882 as not much more than the stainable substance of the cell nucleus. Flemming named this substance according to the Greek word “chroma”, meaning color. In 1911 chromatin was characterized as proteins, named histones, that

  11. Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

    Science.gov (United States)

    Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

    2015-01-01

    mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

  12. Sleep alterations in mammals: did aquatic conditions inhibit rapid eye movement sleep?

    Science.gov (United States)

    Madan, Vibha; Jha, Sushil K

    2012-12-01

    Sleep has been studied widely in mammals and to some extent in other vertebrates. Higher vertebrates such as birds and mammals have evolved an inimitable rapid eye movement (REM) sleep state. During REM sleep, postural muscles become atonic and the temperature regulating machinery remains suspended. Although REM sleep is present in almost all the terrestrial mammals, the aquatic mammals have either radically reduced or completely eliminated REM sleep. Further, we found a significant negative correlation between REM sleep and the adaptation of the organism to live on land or in water. The amount of REM sleep is highest in terrestrial mammals, significantly reduced in semi-aquatic mammals and completely absent or negligible in aquatic mammals. The aquatic mammals are obligate swimmers and have to surface at regular intervals for air. Also, these animals live in thermally challenging environments, where the conductive heat loss is approximately ~90 times greater than air. Therefore, they have to be moving most of the time. As an adaptation, they have evolved unihemispheric sleep, during which they can rove as well as rest. A condition that immobilizes muscle activity and suspends the thermoregulatory machinery, as happens during REM sleep, is not suitable for these animals. It is possible that, in accord with Darwin's theory, aquatic mammals might have abolished REM sleep with time. In this review, we discuss the possibility of the intrinsic role of aquatic conditions in the elimination of REM sleep in the aquatic mammals.

  13. Increases in myocardial workload induced by rapid atrial pacing trigger alterations in global metabolism.

    Directory of Open Access Journals (Sweden)

    Aslan T Turer

    Full Text Available To determine whether increases in cardiac work lead to alterations in the plasma metabolome and whether such changes arise from the heart or peripheral organs.There is growing evidence that the heart influences systemic metabolism through endocrine effects and affecting pathways involved in energy homeostasis.Nineteen patients referred for cardiac catheterization were enrolled. Peripheral and selective coronary sinus (CS blood sampling was performed at serial timepoints following the initiation of pacing, and metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS.Pacing-stress resulted in a 225% increase in the median rate·pressure product from baseline. Increased myocardial work induced significant changes in the peripheral concentration of 43 of 125 metabolites assayed, including large changes in purine [adenosine (+99%, p = 0.006, ADP (+42%, p = 0.01, AMP (+79%, p = 0.004, GDP (+69%, p = 0.003, GMP (+58%, p = 0.01, IMP (+50%, p = 0.03, xanthine (+61%, p = 0.0006], and several bile acid metabolites. The CS changes in metabolites qualitatively mirrored those in the peripheral blood in both timing and magnitude, suggesting the heart was not the major source of the metabolite release.Isolated increases in myocardial work can induce changes in the plasma metabolome, but these changes do not appear to be directly cardiac in origin. A number of these dynamic metabolites have known signaling functions. Our study provides additional evidence to a growing body of literature on metabolic 'cross-talk' between the heart and other organs.

  14. Optical tweezers stretching of chromatin

    NARCIS (Netherlands)

    Pope, L.H.; Bennink, Martin L.; Greve, Jan

    2003-01-01

    Recently significant success has emerged from exciting research involving chromatin stretching using optical tweezers. These experiments, in which a single chromatin fibre is attached by one end to a micron-sized bead held in an optical trap and to a solid surface or second bead via the other end,

  15. Titration and hysteresis in epigenetic chromatin silencing

    International Nuclear Information System (INIS)

    Dayarian, Adel; Sengupta, Anirvan M

    2013-01-01

    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. (paper)

  16. Higher order chromatin organization in cancer

    Science.gov (United States)

    Reddy, Karen L.; Feinberg, Andrew P.

    2013-01-01

    In spite of our increased understanding of how genomes are dysregulated in cancer and a plethora of molecular diagnostic tools, the front line and ‘gold standard’ detection of cancer remains the pathologist’s detection of gross changes in cellular and tissue structure, most strikingly nuclear dis-organization. In fact, for over 140 years it has been noted that nuclear morphology is often disrupted in cancer. Even today, nuclear morphology measures include nuclear size, shape, DNA content (ploidy) and ‘chromatin organization’. Given the importance of nuclear shape to diagnoses of cancer phenotypes, it is surprising and frustrating that we currently lack a detailed understanding to explain these changes and how they might arise and relate to molecular events in the cell. It is an implicit hypothesis that perturbation of chromatin and epigenetic signatures may lead to alterations in nuclear structure (or vice versa) and that these perturbations lie at the heart of cancer genesis. In this review, we attempt to synthesize research leading to our current understanding on how chromatin interactions at the nuclear lamina, epigenetic modulation and gene regulation may intersect in cancer and offer a perspective on critical experiments that would help clarify how nuclear architecture may contribute to the cancerous phenotype. We also discuss the historical understanding of nuclear structure in normal cells and as a diagnostic in cancer. PMID:23266653

  17. Epigenetic regulation of open chromatin in pluripotent stem cells

    Science.gov (United States)

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2014-01-01

    The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues that had been unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, eventually alter the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is globally open to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells. This includes microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genome-wide nucleosome accessibility and nucleosome positioning. Further understanding of the epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem. PMID:24695097

  18. DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

    Science.gov (United States)

    Rother, Magdalena B; van Attikum, Haico

    2017-10-05

    Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

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

    Science.gov (United States)

    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.

  20. Na+ Influx Induced by New Antimalarials Causes Rapid Alterations in the Cholesterol Content and Morphology of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Sudipta Das

    2016-05-01

    Full Text Available Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2, glycosylphosphotidylinositol (GPI-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble "rhoptries" and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise.

  1. RNA is an integral component of chromatin that contributes to its structural organization.

    Directory of Open Access Journals (Sweden)

    Antonio Rodríguez-Campos

    Full Text Available Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA(- and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with alpha-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

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

    Science.gov (United States)

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

    2006-06-01

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

  3. Alteration of chromophoric dissolved organic matter by solar UV radiation causes rapid changes in bacterial community composition†

    Science.gov (United States)

    Piccini, Claudia; Conde, Daniel; Pernthaler, Jakob; Sommaruga, Ruben

    2010-01-01

    We evaluated the effect of photochemical alterations of chromophoric dissolved organic matter (CDOM) on bacterial abundance, activity and community composition in a coastal lagoon of the Atlantic Ocean with high dissolved organic carbon concentration. On two occasions during the austral summer, bacteria-free water of the lagoon was exposed to different regions of the solar spectrum (full solar radiation, UV-A + PAR, PAR) or kept in the dark. Subsequently, dilution cultures were established with bacterioplankton from the lagoon that were incubated in the pre-exposed water for 5 h in the dark. Cell abundance, activity, and community composition of bacterioplankton were assessed before and after incubation in the different treatments. Changes in absorption, fluorescence, and DOC concentration were used as proxies for CDOM photoalteration. We found a significant CDOM photobleaching signal, DOC loss, as well as a stimulation of bacterial activity in the treatments pre-exposed to UV radiation, suggesting increased bioavailability of DOM. Bacterial community analysis by fluorescence in situ hybridization revealed that this stimulation was mainly accompanied by the specific enrichment of Alpha- and Betaproteobacteria. Thus, our results suggest that CDOM photoalteration not only stimulates bacterioplankton growth, but also induces rapid changes in bacterioplankton composition, which can be of relevance for ecosystem functioning, particularly considering present and future changes in the input of terrestrial CDOM to aquatic systems. PMID:19707620

  4. Alteration of chromophoric dissolved organic matter by solar UV radiation causes rapid changes in bacterial community composition.

    Science.gov (United States)

    Piccini, Claudia; Conde, Daniel; Pernthaler, Jakob; Sommaruga, Ruben

    2009-09-01

    We evaluated the effect of photochemical alterations of chromophoric dissolved organic matter (CDOM) on bacterial abundance, activity and community composition in a coastal lagoon of the Atlantic Ocean with high dissolved organic carbon concentration. On two occasions during the austral summer, bacteria-free water of the lagoon was exposed to different regions of the solar spectrum (full solar radiation, UV-A+PAR, PAR) or kept in the dark. Subsequently, dilution cultures were established with bacterioplankton from the lagoon that were incubated in the pre-exposed water for 5 h in the dark. Cell abundance, activity, and community composition of bacterioplankton were assessed before and after incubation in the different treatments. Changes in absorption, fluorescence, and DOC concentration were used as proxies for CDOM photoalteration. We found a significant CDOM photobleaching signal, DOC loss, as well as a stimulation of bacterial activity in the treatments pre-exposed to UV radiation, suggesting increased bioavailability of DOM. Bacterial community analysis by fluorescence in situ hybridization revealed that this stimulation was mainly accompanied by the specific enrichment of Alpha- and Betaproteobacteria. Thus, our results suggest that CDOM photoalteration not only stimulates bacterioplankton growth, but also induces rapid changes in bacterioplankton composition, which can be of relevance for ecosystem functioning, particularly considering present and future changes in the input of terrestrial CDOM to aquatic systems.

  5. Oxidative stress signaling to chromatin in health and disease

    KAUST Repository

    Kreuz, Sarah

    2016-06-20

    Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation.

  6. Chromatin versus pathogens: the function of epigenetics in plant immunity

    Science.gov (United States)

    Ding, Bo; Wang, Guo-Liang

    2015-01-01

    To defend against pathogens, plants have developed a sophisticated innate immunity that includes effector recognition, signal transduction, and rapid defense responses. Recent evidence has demonstrated that plants utilize the epigenetic control of gene expression to fine-tune their defense when challenged by pathogens. In this review, we highlight the current understanding of the molecular mechanisms of histone modifications (i.e., methylation, acetylation, and ubiquitination) and chromatin remodeling that contribute to plant immunity against pathogens. Functions of key histone-modifying and chromatin remodeling enzymes are discussed. PMID:26388882

  7. Rapid intravenous infusion of 20 mL/kg saline alters the distribution of perfusion in healthy supine humans.

    Science.gov (United States)

    Henderson, A C; Sá, R C; Barash, I A; Holverda, S; Buxton, R B; Hopkins, S R; Prisk, G K

    2012-03-15

    Rapid intravenous saline infusion, a model meant to replicate the initial changes leading to pulmonary interstitial edema, increases pulmonary arterial pressure in humans. We hypothesized that this would alter lung perfusion distribution. Six healthy subjects (29 ± 6 years) underwent magnetic resonance imaging to quantify perfusion using arterial spin labeling. Regional proton density was measured using a fast-gradient echo sequence, allowing blood delivered to the slice to be normalized for density and quantified in mL/min/g. Contributions from flow in large conduit vessels were minimized using a flow cutoff value (blood delivered > 35% maximum in mL/min/cm(3)) in order to obtain an estimate of blood delivered to the capillary bed (perfusion). Images were acquired supine at baseline, after infusion of 20 mL/kg saline, and after a short upright recovery period for a single sagittal slice in the right lung during breath-holds at functional residual capacity. Thoracic fluid content measured by impedance cardiography was elevated post-infusion by up to 13% (pchanges in conduit vessels, there were no significant changes in perfusion in dependent lung following infusion (7.8 ± 1.9 mL/min/g baseline, 7.9 ± 2.0 post, 8.5 ± 2.1 recovery, p=0.36). There were no significant changes in lung density. These data suggest that saline infusion increased perfusion to nondependent lung, consistent with an increase in intravascular pressures. Dependent lung may have been "protected" from increases in perfusion following infusion due to gravitational compression of the pulmonary vasculature. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Chromatin Remodeling and Plant Immunity.

    Science.gov (United States)

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

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance? © 2017 Elsevier Inc. All rights reserved.

  9. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

  10. The effect of higher order chromatin structure on DNA damage and repair

    International Nuclear Information System (INIS)

    Yasui, L.S.; Warters, R.L.; Higashikubo, R.

    1985-01-01

    Alterations in chromatin structure are thought to play an important role in various radiobiological end points, i.e., DNA damage, DNA damage repair and cell survival. The authors use here the isoleucine deprivation technique to decondense higher order chromatin structure and asses X-ray induced DNA damage, DNA damage repair and cell survival on cells with decondensed chromatin as compared to controls. This chromatin decondensation manifests itself as a 30 fold decrease in nuclear area occupied by heterochromatin, an increased rate of Micrococcal nuclease digestion, 15% increased ethidium bromide intercalation and an altered binding capacity of Hl histone. These chromatin/nuclear changes do not affect X-ray induced DNA damage as measured by the alkaline elution technique or cell survival but slows DNA damage repair by 2 fold. Therefore, even though the chromatin appears more accessible to DNA damage and repair processes, these particular nuclear changes do not affect the DNA damaging effects of X-rays and in addition, repair is not enhanced by the ''relaxed'' state of chromatin. It is proposed that the altered metabolic state of isoleucine deprived cells provides a less efficient system for the repair of X-ray induced DNA damage

  11. High-Frequency Promoter Firing Links THO Complex Function to Heavy Chromatin Formation

    DEFF Research Database (Denmark)

    Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu

    2013-01-01

    The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes...... (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single......-molecule fluorescence insitu hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase...

  12. A Long-Distance Chromatin Affair

    NARCIS (Netherlands)

    Denker, Annette; de Laat, Wouter

    2015-01-01

    Changes in transcription factor binding sequences result in correlated changes in chromatin composition locally and at sites hundreds of kilobases away. New studies demonstrate that this concordance is mediated via spatial chromatin interactions that constitute regulatory modules of the human

  13. Guarding against Collateral Damage during Chromatin Transactions

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Lukas, Jiri

    2013-01-01

    Signal amplifications are vital for chromatin function, yet they also bear the risk of transforming into unrestrained, self-escalating, and potentially harmful responses. Examples of inbuilt limitations are emerging, revealing how chromatin transactions are confined within physiological boundaries....

  14. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Persson, Jenna [Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet (Sweden); Ekwall, Karl, E-mail: karl.ekwall@ki.se [Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet (Sweden); School of Life Sciences, University College Sodertorn, NOVUM, Huddinge (Sweden)

    2010-05-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  15. Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light

    International Nuclear Information System (INIS)

    Strniste, G.F.; Rall, S.C.

    1976-01-01

    Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; a Sepharose 4B column assay in which protein eluted coincident with DNA; and a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein--DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure

  16. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    International Nuclear Information System (INIS)

    Persson, Jenna; Ekwall, Karl

    2010-01-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  17. Circular chromatin complexes in human lymphocytes high-resolution autoradiography

    International Nuclear Information System (INIS)

    Becak, M.L.; Fukuda-Pizzocaro, K.; Santos, R. de C.S. dos; Brunner, O.

    1985-01-01

    Transcriptionally active chromatin fibers were observed in chromosomes presenting the loops/scaffold configuration. The active fibers showed altered nucleosomes and presented multiforked aspects which led to the formation of ring complexes. The ribonucleoprotein transcripts (RNP) appeared as networks of 0.1 μm or multiples tandemly disposed along the fiber. It is suggested that the ring complexes belong to the human genome. The possibility that these circular structures come from a prokaryote is also considered. (author) [pt

  18. Excision of x-ray-induced thymine damage in chromatin from heated cells

    International Nuclear Information System (INIS)

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

    1979-01-01

    Experiments were performed to distinguish between two possible modes of hyperthermia-induced inhibition of thymine base damage excision from the DNA of CHO cells: (1) heat denaturation of excision enzyme(s) or (2) heat-induced alteration of the substrate for damage excision (chromatin). While hyperthermia (45 0 C, 15 min) had no apparent effect on the capacity of the excision enzymes to excise damage from DNA it had a dramatic effect (ca. 80% inhibition) on the ability of chromatin to serve as a substrate for unheated enzymes. These results suggest that hyperthermia-induced radiosensitization of CHO cells may be due primarily to lesions in the cellular chromatin

  19. Early aberrations in chromatin dynamics in embryos produced under In vitro conditions

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Strejcek, Frantisek

    2012-01-01

    standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin...... decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar...

  20. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Hierarchical role for transcription factors and chromatin structure in genome organization along adipogenesis

    DEFF Research Database (Denmark)

    Sarusi Portuguez, Avital; Schwartz, Michal; Siersbaek, Rasmus

    2017-01-01

    The three dimensional folding of mammalian genomes is cell type specific and difficult to alter suggesting that it is an important component of gene regulation. However, given the multitude of chromatin-associating factors, the mechanisms driving the colocalization of active chromosomal domains...... by PPARγ and Lpin1, undergoes orchestrated reorganization during adipogenesis. Coupling the dynamics of genome architecture with multiple chromatin datasets indicated that among all the transcription factors (TFs) tested, RXR is central to genome reorganization at the beginning of adipogenesis...

  2. Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: Biological and clinical significance

    International Nuclear Information System (INIS)

    Terzoudi, Georgia I.; Hatzi, Vasiliki I.; Donta-Bakoyianni, Catherine; Pantelias, Gabriel E.

    2011-01-01

    The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.

  3. Chromatin-bound RNA and the neurobiology of psychiatric disease.

    Science.gov (United States)

    Tushir, J S; Akbarian, S

    2014-04-04

    A large, and still rapidly expanding literature on epigenetic regulation in the nervous system has provided fundamental insights into the dynamic regulation of DNA methylation and post-translational histone modifications in the context of neuronal plasticity in health and disease. Remarkably, however, very little is known about the potential role of chromatin-bound RNAs, including many long non-coding transcripts and various types of small RNAs. Here, we provide an overview on RNA-mediated regulation of chromatin structure and function, with focus on histone lysine methylation and psychiatric disease. Examples of recently discovered chromatin-bound long non-coding RNAs important for neuronal health and function include the brain-derived neurotrophic factor antisense transcript (Bdnf-AS) which regulates expression of the corresponding sense transcript, and LOC389023 which is associated with human-specific histone methylation signatures at the chromosome 2q14.1 neurodevelopmental risk locus by regulating expression of DPP10, an auxillary subunit for voltage-gated K(+) channels. We predict that the exploration of chromatin-bound RNA will significantly advance our current knowledge base in neuroepigenetics and biological psychiatry. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Regulation of chromatin structure by poly(ADP-ribosylation

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

    Full Text Available 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.

  5. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

    Science.gov (United States)

    Rahiminia, Tahereh; Hosseini, Akram; Anvari, Morteza; Ghasemi-Esmailabad, Saeed; Talebi, Ali Reza

    2017-08-01

    Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome

  6. Chromatin dynamics during DSB repair

    Czech Academy of Sciences Publication Activity Database

    Falk, Martin; Lukášová, Emilie; Gabrielová, Barbora; Ondřej, Vladan; Kozubek, Stanislav

    2007-01-01

    Roč. 1773, č. 10 (2007), s. 1534-1545 ISSN 0167-4889 R&D Projects: GA ČR(CZ) GP204/06/P349; GA ČR(CZ) 1QS500040508; GA AV ČR(CZ) IAA1065203; GA MŠk(CZ) 1P05OC084 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : chromatin structure * double- strand breaks (DSB) * DNA repair Subject RIV: BO - Biophysics Impact factor: 4.374, year: 2007

  7. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    Adipocyte differentiation is tightly controlled by a transcriptional cascade, which directs the extensive reprogramming of gene expression required to convert fibroblast-like precursor cells into mature lipid-laden adipocytes. Recent global analyses of transcription factor binding and chromatin...... remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications....... Such transcription factor hotspots are likely to represent key signaling nodes which integrate multiple adipogenic signals at specific chromatin sites, thereby facilitating coordinated action on gene expression....

  8. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of

  9. A microscopic analysis of Arabidopsis chromatin

    NARCIS (Netherlands)

    Willemse, J.J.

    2007-01-01

    Genetic information of eukaryotic organisms is stored as DNA in the nuclei of their cells. Nuclear DNA is associated with several proteins, which together form chromatin. The most abundant chromatin proteins arehistones,they arrange the initial packaging step of the DNA. DNA

  10. Chromatin dynamics resolved with force spectroscopy

    NARCIS (Netherlands)

    Chien, Fan-Tso

    2011-01-01

    In eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is connected to the adjacent nucleosomes with linker DNA. The folding of chromatin fibers effectively increases

  11. Transcription Through Chromatin - Dynamic Organization of Genes

    Indian Academy of Sciences (India)

    different proteins involved in the synthesis of mRNA from the. DNA template. ... CBP - CREB Binding Protein. CHRAC. Chromatin .... nucleosomal interactions, and thereby change the chromatin structure, as per the ..... methyltransferases in gene regulation is yet to be elucidated. .... Molecular Biology and. Genetics Unit.

  12. Chromatin Remodelers: From Function to Dysfunction

    Directory of Open Access Journals (Sweden)

    Gernot Längst

    2015-06-01

    Full Text Available 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.

  13. Rapid increases in permeability and porosity of bentonite-sand mixtures due to alteration by water vapor

    International Nuclear Information System (INIS)

    Couture, R.A.

    1984-01-01

    Packed columns of canister packing material containing 25% bentonite and 75% quartz or basalt sand, were exposed to water vapor at temperatures up t 260 0 C. The permeabilities of the columns were subsequently measured after complete saturation with liquid water in a pressurized system. Exposure to water vapor caused irreversible increases in permeability by factors of up to 10 5 . After saturation with liquid water, the permeability was nearly independent of temperature. The increases in permeability were due to a large decrease in the ability of the bentonite to swell in water. Calculations suggest that swelling of bentonite altered at 250 0 C was not sufficient to fill the pore spaces. If the pore spaces are filled, the mixture will form an effective barrier against flow, diffusion, and transport of colloids. The results suggest that if bentonite-based canister packing material is exposed even briefly to water vapor at high temperatures in a high-level nuclear waste repository, its performance will be seriously impaired. The problem is less severe if the proportion of bentonite is high and the material is highly compacted. Previous results show significant degradation of bentonite by water vapor at temperatures as low as 150 0 C. This suggests that in some repositories, backfill in tunnels and drifts may also be affected. 9 references, 5 figures, 1 table

  14. Rapid Genetic and Epigenetic Alterations under Intergeneric Genomic Shock in Newly Synthesized Chrysanthemum morifolium × Leucanthemum paludosum Hybrids (Asteraceae)

    Science.gov (United States)

    Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Fang, Weimin; Guan, Zhiyong; Teng, Nianjun; Liao, Yuan; Chen, Fadi

    2014-01-01

    The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5–50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses. PMID:24407856

  15. Rapid genetic and epigenetic alterations under intergeneric genomic shock in newly synthesized Chrysanthemum morifolium x Leucanthemum paludosum hybrids (Asteraceae).

    Science.gov (United States)

    Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Fang, Weimin; Guan, Zhiyong; Teng, Nianjun; Liao, Yuan; Chen, Fadi

    2014-01-01

    The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5-50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.

  16. Rapid modulation of ultraviolet shielding in plants is influenced by solar ultraviolet radiation and linked to alterations in flavonoids.

    Science.gov (United States)

    Barnes, Paul W; Tobler, Mark A; Keefover-Ring, Ken; Flint, Stephan D; Barkley, Anne E; Ryel, Ronald J; Lindroth, Richard L

    2016-01-01

    The accumulation of ultraviolet (UV)-absorbing compounds (flavonoids and related phenylpropanoids) and the resultant decrease in epidermal UV transmittance (TUV ) are primary protective mechanisms employed by plants against potentially damaging solar UV radiation and are critical components of the overall acclimation response of plants to changing solar UV environments. Whether plants can adjust this UV sunscreen protection in response to rapid changes in UV, as occurs on a diurnal basis, is largely unexplored. Here, we use a combination of approaches to demonstrate that plants can modulate their UV-screening properties within minutes to hours, and these changes are driven, in part, by UV radiation. For the cultivated species Abelmoschus esculentus, large (30-50%) and reversible changes in TUV occurred on a diurnal basis, and these adjustments were associated with changes in the concentrations of whole-leaf UV-absorbing compounds and several quercetin glycosides. Similar results were found for two other species (Vicia faba and Solanum lycopersicum), but no such changes were detected in Zea mays. These findings reveal a much more dynamic UV-protection mechanism than previously recognized, raise important questions concerning the costs and benefits of UV-protection strategies in plants and have practical implications for employing UV to enhance crop vigor and quality in controlled environments. © 2015 John Wiley & Sons Ltd.

  17. Chromatin changes in response to drought, salinity, heat, and cold stresses in plants

    Directory of Open Access Journals (Sweden)

    Jong-Myong eKim

    2015-03-01

    Full Text Available Chromatin regulation is essential to regulate genes and genome activities. In plants, the alteration of histone modification and DNA methylation are coordinated with changes in the expression of stress-responsive genes to adapt to environmental changes. Several chromatin regulators have been shown to be involved in the regulation of stress-responsive gene networks under abiotic stress conditions. Specific histone modification sites and the histone modifiers that regulate key stress-responsive genes have been identified by genetic and biochemical approaches, revealing the importance of chromatin regulation in plant stress responses. Recent studies have also suggested that histone modification plays an important role in plant stress memory. In this review, we summarize recent progress on the regulation and alteration of histone modification (acetylation, methylation, phosphorylation, and SUMOylation in response to the abiotic stresses, drought, high-salinity, heat, and cold in plants.

  18. Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury.

    Science.gov (United States)

    Züchner, Mark; Kondratskaya, Elena; Sylte, Camilla B; Glover, Joel C; Boulland, Jean-Luc

    2018-01-15

    were two main locomotor frequencies, but injured spinal cords exhibited a shift towards the higher frequency. Injury also altered the neurochemical dependence of locomotor CPG output, such that injured spinal cords, unlike control spinal cords, were incapable of generating low frequency rhythmic coordinated activity in the presence of NMDA and dopamine alone. Thus, the neonatal spinal cord also exhibits remarkable functional recovery after lumbar injuries, but the neurochemical sensitivity of locomotor circuitry is modified in the process. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  19. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

    Science.gov (United States)

    Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

    2016-01-01

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

  20. Structured illumination to spatially map chromatin motions.

    Science.gov (United States)

    Bonin, Keith; Smelser, Amanda; Moreno, Naike Salvador; Holzwarth, George; Wang, Kevin; Levy, Preston; Vidi, Pierre-Alexandre

    2018-05-01

    We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340  ±  30  nm, which simultaneously photoactivate a 7  ×  7 matrix pattern of GFP-labeled histones, with spots 1.70  μm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  1. Extensive Variation in Chromatin States Across Humans

    KAUST Repository

    Kasowski, M.

    2013-10-17

    The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

  2. Extensive Variation in Chromatin States Across Humans

    KAUST Repository

    Kasowski, M.; Kyriazopoulou-Panagiotopoulou, S.; Grubert, F.; Zaugg, J. B.; Kundaje, A.; Liu, Y.; Boyle, A. P.; Zhang, Q. C.; Zakharia, F.; Spacek, D. V.; Li, J.; Xie, D.; Olarerin-George, A.; Steinmetz, L. M.; Hogenesch, J. B.; Kellis, M.; Batzoglou, S.; Snyder, M.

    2013-01-01

    The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

  3. Effects of Optogenetic inhibition of BLA on Sleep Brief Optogenetic Inhibition of the Basolateral Amygdala in Mice Alters Effects of Stressful Experiences on Rapid Eye Movement Sleep.

    Science.gov (United States)

    Machida, Mayumi; Wellman, Laurie L; Fitzpatrick Bs, Mairen E; Hallum Bs, Olga; Sutton Bs, Amy M; Lonart, György; Sanford, Larry D

    2017-04-01

    Stressful events can directly produce significant alterations in subsequent sleep, in particular rapid eye movement sleep (REM); however, the neural mechanisms underlying the process are not fully known. Here, we investigated the role of the basolateral nuclei of the amygdala (BLA) in regulating the effects of stressful experience on sleep. We used optogenetics to briefly inhibit glutamatergic cells in BLA during the presentation of inescapable footshock (IS) and assessed effects on sleep, the acute stress response, and fear memory. c-Fos expression was also assessed in the amygdala and the medial prefrontal cortex (mPFC), both regions involved in coping with stress, and in brain stem regions implicated in the regulation of REM. Compared to control mice, peri-shock inhibition of BLA attenuated an immediate reduction in REM after IS and produced a significant overall increase in REM. Moreover, upon exposure to the shock context alone, mice receiving peri-shock inhibition of BLA during training showed increased REM without altered freezing (an index of fear memory) or stress-induced hyperthermia (an index of acute stress response). Inhibition of BLA during REM under freely sleeping conditions enhanced REM only when body temperature was high, suggesting the effect was influenced by stress. Peri-shock inhibition of BLA also led to elevated c-Fos expression in the central nucleus of the amygdala and mPFC and differentially altered c-Fos activity in the selected brain stem regions. Glutamatergic cells in BLA can modulate the effects of stress on REM and can mediate effects of fear memory on sleep that can be independent of behavioral fear. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  4. Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

    Science.gov (United States)

    Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have

  5. Altered composition of bone as triggered by irradiation facilitates the rapid erosion of the matrix by both cellular and physicochemical processes.

    Directory of Open Access Journals (Sweden)

    Danielle E Green

    Full Text Available Radiation rapidly undermines trabecular architecture, a destructive process which proceeds despite a devastated cell population. In addition to the 'biologically orchestrated' resorption of the matrix by osteoclasts, physicochemical processes enabled by a damaged matrix may contribute to the rapid erosion of bone quality. 8w male C57BL/6 mice exposed to 5 Gy of Cs(137 γ-irradiation were compared to age-matched control at 2d, 10d, or 8w following exposure. By 10d, irradiation had led to significant loss of trabecular bone volume fraction. Assessed by reflection-based Fourier transform infrared imaging (FTIRI, chemical composition of the irradiated matrix indicated that mineralization had diminished at 2d by -4.3±4.8%, and at 10d by -5.8±3.2%. These data suggest that irradiation facilitates the dissolution of the matrix through a change in the material itself, a conclusion supported by a 13.7±4.5% increase in the elastic modulus as measured by nanoindentation. The decline in viable cells within the marrow of irradiated mice at 2d implies that the immediate collapse of bone quality and inherent increased risk of fracture is not solely a result of an overly-active biologic process, but one fostered by alterations in the material matrix that predisposes the material to erosion.

  6. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

    2014-01-01

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  7. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  8. Chromatin Pioneers | Center for Cancer Research

    Science.gov (United States)

    Taking advantage of their ability to explore provocative ideas, NCI investigators pioneered the study of chromatin to demonstrate its functional importance and lay the groundwork for understanding its role in cancer and other diseases.

  9. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja

    2009-01-01

    Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...

  10. High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

    Science.gov (United States)

    Rosa-Garrido, Manuel; Chapski, Douglas J; Schmitt, Anthony D; Kimball, Todd H; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J; Ren, Shuxun; Wang, Yibin; Ren, Bing; Vondriska, Thomas M

    2017-10-24

    Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. © 2017 The Authors.

  11. Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

    International Nuclear Information System (INIS)

    Cherkezyan, Lusik; Backman, Vadim; Stypula-Cyrus, Yolanda; Subramanian, Hariharan; White, Craig; Dela Cruz, Mart; Wali, Ramesh K; Goldberg, Michael J; Bianchi, Laura K; Roy, Hemant K

    2014-01-01

    Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention. For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function. We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential. We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well

  12. Dynamic Rewiring of Promoter-Anchored Chromatin Loops during Adipocyte Differentiation

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Madsen, Jesper Grud Skat; Javierre, Biola Maria

    2017-01-01

    -C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced...

  13. H2O2-induced higher order chromatin degradation: A novel ...

    Indian Academy of Sciences (India)

    Unknown

    mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic ... clease works through a single strand scission mechanism ... a great mutagenic risk to the surviving cells, because en-.

  14. Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.

    Science.gov (United States)

    Amemiya, Kenji; Hirotsu, Yosuke; Goto, Taichiro; Nakagomi, Hiroshi; Mochizuki, Hitoshi; Oyama, Toshio; Omata, Masao

    2016-12-01

    Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time-consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]-seq), to detect somatic mutations in tumors. We prepared FFPE tissues and TIC specimens from tumors in nine lung cancer patients and one patient with breast cancer. We found that the quality and quantity of TIC DNA was higher than that of FFPE DNA, which requires microdissection to enrich DNA from target tissues. Targeted sequencing using a next-generation sequencer obtained sufficient sequence data using TIC DNA. Most (92%) somatic mutations in lung primary tumors were found to be consistent between TIC and FFPE DNA. We also applied TIC DNA to primary and metastatic tumor tissues to analyze tumor heterogeneity in a breast cancer patient, and showed that common and distinct mutations among primary and metastatic sites could be classified into two distinct histological subtypes. TIC-seq is an alternative and feasible method to analyze genomic alterations in tumors by simply touching the cut surface of specimens to slides. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  15. FGF signalling regulates chromatin organisation during neural differentiation via mechanisms that can be uncoupled from transcription.

    Directory of Open Access Journals (Sweden)

    Nishal S Patel

    Full Text Available Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR signalling in Raldh2-/- embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that

  16. RegulatING chromatin regulators

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Nabbi, Arash; Riabowol, Karl

    2013-01-01

    The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on...

  17. ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

    Science.gov (United States)

    Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

    2017-10-05

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  18. Sequential chromatin immunoprecipitation to detect SUMOylated MeCP2 in neurons

    Directory of Open Access Journals (Sweden)

    Tao Wu

    2016-03-01

    Full Text Available The small ubiquitin-like modifier (SUMO is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM. Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2 is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.

  19. Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation.

    Science.gov (United States)

    Gao, Lexuan; Geng, Yupeng; Li, Bo; Chen, Jiakuan; Yang, Ji

    2010-11-01

    Alternanthera philoxeroides (alligator weed) is an invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation in its introduced range. The mechanisms underpinning the wide range of phenotypic variation and rapid adaptation to novel and changing environments remain uncharacterized. In this study, we examined the epigenetic variation and its correlation with phenotypic variation in plants exposed to natural and manipulated environmental variability. Genome-wide methylation profiling using methylation-sensitive amplified fragment length polymorphism (MSAP) revealed considerable DNA methylation polymorphisms within and between natural populations. Plants of different source populations not only underwent significant morphological changes in common garden environments, but also underwent a genome-wide epigenetic reprogramming in response to different treatments. Methylation alterations associated with response to different water availability were detected in 78.2% (169/216) of common garden induced polymorphic sites, demonstrating the environmental sensitivity and flexibility of the epigenetic regulatory system. These data provide evidence of the correlation between epigenetic reprogramming and the reversible phenotypic response of alligator weed to particular environmental factors. © 2010 Blackwell Publishing Ltd.

  20. Altering the cooling rate dependence of phase formation during rapid solidification in the Nd{sub 2}Fe{sub 14}B system

    Energy Technology Data Exchange (ETDEWEB)

    Branagan, D.J. [USDOE, Ames, IA (United States). Ames Lab.]|[Iowa State Univ. of Science and Technology, Ames, IA (United States). Dept. of Materials Science and Engineering; McCallum, R.W. [USDOE, Ames, IA (United States). Ames Lab.]|[Iowa State Univ. of Science and Technology, Ames, IA (United States). Dept. of Materials Science and Engineering

    1995-04-26

    In order to evaluate the effects of additions on the solidification behavior of Nd{sub 2}Fe{sub 14}B, a stoichiometric alloy was modified with elemental additions of Ti or C and a compound addition of Ti with C. For each alloy, a series of wheel speed runs was undertaken, from which the optimum wheel speeds and optimum energy products were determined. On the BH{sub max} versus wheel speed plots, regions were identified in order to analyze the changes with cooling rates leading to phase formation brought about by the alloy modifications. The compilation of the regional data of the modified alloys showed their effects on altering the cooling rate dependence of phase formation. It was found that the regions of properitectic iron formation, glass formation, and the optimum cooling rate can be changed by more than a factor of two through appropriate alloying additions. The effects of the alloy modifications can be visualized in a convenient fashion through the use of a model continuous cooling transformation (CCT) diagram which represents phase formation during the solidification process under continuous cooling conditions for a wide range of cooling rates from rapid solidification to equilibrium cooling. ((orig.)).

  1. Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

    2018-03-01

    Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Not just gene expression: 3D implications of chromatin modifications during sexual plant reproduction.

    Science.gov (United States)

    Dukowic-Schulze, Stefanie; Liu, Chang; Chen, Changbin

    2018-01-01

    DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.

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

    Science.gov (United States)

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

    2014-09-29

    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.

  4. ATP-dependent chromatin remodeling in the DNA-damage response

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  5. Neutron-scattering studies of chromatin

    International Nuclear Information System (INIS)

    Bradbury, E.M.; Baldwin, J.P.; Carpenter, B.G.; Hjelm, R.P.; Hancock, R.; Ibel, K.

    1976-01-01

    It is clear that a knowledge of the basic molecular structure of chromatin is a prerequisite for any progress toward an understanding of chromosome organization. With a two-component system, protein and nucleic acid, neutrons have a particularly powerful application to studies of the spatial arrangements of these components because of the ability, by contrast matching with H 2 O-D 2 O mixtures, to obtain neutron-scattering data on the individual components. With this approach it has been shown that the neutron diffraction of chromatin is consistent with a ''beads on a string'' model in which the bead consists of a protein core with DNA coiled on the outside. However, because chromatin is a gel and gives limited structural data, confirmation of such a model requires extension of the neutron studies by deuteration of specific chromatin components and the isolation of chromatin subunits. Although these studies are not complete, the neutron results so far obtained support the subunit model described above

  6. New mitotic regulators released from chromatin

    Directory of Open Access Journals (Sweden)

    Hideki eYokoyama

    2013-12-01

    Full Text Available Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP, produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP.

  7. Map of open and closed chromatin domains in Drosophila genome.

    Science.gov (United States)

    Milon, Beatrice; Sun, Yezhou; Chang, Weizhong; Creasy, Todd; Mahurkar, Anup; Shetty, Amol; Nurminsky, Dmitry; Nurminskaya, Maria

    2014-11-18

    Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse "exceptions" from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin. These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.

  8. A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development

    Directory of Open Access Journals (Sweden)

    Justyna Nitarska

    2016-11-01

    Full Text Available Histone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.

  9. Condensins Exert Force on Chromatin-Nuclear Envelope Tethers to Mediate Nucleoplasmic Reticulum Formation in Drosophila melanogaster

    Science.gov (United States)

    Bozler, Julianna; Nguyen, Huy Q.; Rogers, Gregory C.; Bosco, Giovanni

    2014-01-01

    Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. PMID:25552604

  10. Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster.

    Science.gov (United States)

    Bozler, Julianna; Nguyen, Huy Q; Rogers, Gregory C; Bosco, Giovanni

    2014-12-30

    Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. Copyright © 2015 Bozler et al.

  11. Autism genes keep turning up chromatin.

    Science.gov (United States)

    Lasalle, Janine M

    2013-06-19

    Autism-spectrum disorders (ASD) are complex genetic disorders collectively characterized by impaired social interactions and language as well as repetitive and restrictive behaviors. Of the hundreds of genes implicated in ASD, those encoding proteins acting at neuronal synapses have been most characterized by candidate gene studies. However, recent unbiased genome-wide analyses have turned up a multitude of novel candidate genes encoding nuclear factors implicated in chromatin remodeling, histone demethylation, histone variants, and the recognition of DNA methylation. Furthermore, the chromatin landscape of the human genome has been shown to influence the location of de novo mutations observed in ASD as well as the landscape of DNA methylation underlying neurodevelopmental and synaptic processes. Understanding the interactions of nuclear chromatin proteins and DNA with signal transduction pathways and environmental influences in the developing brain will be critical to understanding the relevance of these ASD candidate genes and continued uncovering of the "roots" of autism etiology.

  12. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

    DEFF Research Database (Denmark)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po

    2014-01-01

    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 3,995 proteins. The replication machinery and 485 chromatin factors...... such as CAF-1, DNMT1 and 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, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment...

  13. CHD chromatin remodelers and the transcription cycle

    Science.gov (United States)

    Murawska, Magdalena

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by “opening” or “closing” chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but also are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts. PMID:22223048

  14. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...... is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation...

  15. Modulation of chromatin access during adipocyte differentiation

    DEFF Research Database (Denmark)

    Mandrup, Susanne; Hager, Gordon L

    2012-01-01

    identified; however, it is not until recently that we have begun to understand how these factors act at a genome-wide scale. In a recent publication we have mapped the genome-wide changes in chromatin structure during differentiation of 3T3-L1 preadipocytes and shown that a major reorganization...... of the chromatin landscape occurs within few hours following the addition of the adipogenic cocktail. In addition, we have mapped the genome-wide profiles of several of the early adipogenic transcription factors and shown that they act in a highly cooperative manner to drive this dramatic remodeling process....

  16. Chromatin and lamin A determine two different mechanical response regimes of the cell nucleus.

    Science.gov (United States)

    Stephens, Andrew D; Banigan, Edward J; Adam, Stephen A; Goldman, Robert D; Marko, John F

    2017-07-07

    The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (<3 μm), and euchromatin/heterochromatin levels modulate the stiffness. In contrast, lamin A/C levels control nuclear strain stiffening at large extensions. These results can be understood through simulations of a polymeric shell and cross-linked polymer interior. Our results provide a framework for understanding the differential effects of chromatin and lamin A/C in cell nuclear mechanics and their alterations in disease. © 2017 Stephens et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

    Science.gov (United States)

    Soto-Feliciano, Yadira M; Bartlebaugh, Jordan M E; Liu, Yunpeng; Sánchez-Rivera, Francisco J; Bhutkar, Arjun; Weintraub, Abraham S; Buenrostro, Jason D; Cheng, Christine S; Regev, Aviv; Jacks, Tyler E; Young, Richard A; Hemann, Michael T

    2017-05-15

    Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6 KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. © 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press.

  18. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    Science.gov (United States)

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Chakraborty, Payal [Bionivid Technology Pvt Ltd, Kasturi Nagar, Bangalore 560043 (India); Jana, Kuladip [Division of Molecular Medicine, Centre for Translational Animal Research, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, West Bengal (India); Das, Chandrima, E-mail: chandrima.das@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Dasgupta, Dipak, E-mail: dipak.dasgupta@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Ashwat Visvanathan

    Full Text Available 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.

  2. Chromatin damage induced by fast neutrons or UV laser radiation

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L.; Constantinescu, B.; Gazdaru, D.; Mihailescu, I

    2002-07-01

    Chromatin samples from livers of Wistar rats were subjected to fast neutron irradiation in doses of 10-100 Gy or to a 248 nm excimer laser radiation, in doses of 0.5-3 MJ.m{sup -2}. The action of the radiation on chromatin was monitored by chromatin intrinsic fluorescence and fluorescence lifetimes (of bound ethidium bromide to chromatin) and by analysing fluorescence resonance energy transfer between dansyl chloride and acridine orange coupled to chromatin. For the mentioned doses of UV excimer laser radiation, the action on chromatin was more intense than in the case of fast neutrons. The same types of damage are produced by the two radiations: acidic and basic destruction of chromatin protein structure, DNA strand breaking and the increase of the distance between DNA and proteins in chromatin. (author)

  3. Classical and Nonclassical Estrogen Receptor Action on Chromatin Templates

    National Research Council Canada - National Science Library

    Nordeen, Steven

    2000-01-01

    .... Using newly-developed approaches, I investigated mechanisms of estrogen/estrogen receptor action on chromatin templates in vitro in order to better understand the role of chromatin in steroid-regulated gene expression...

  4. Classical and Nonclassical Estrogen Receptor Action on Chromatin Templaces

    National Research Council Canada - National Science Library

    Nordeen, Steve

    2001-01-01

    .... Using newly-developed approaches, I investigated mechanisms of estrogen/estrogen receptor action on chromatin templates in vitro in order to better understand the role of chromatin in steroid-regulated gene expression...

  5. Chromatin Repressive Complexes in Stem Cells, Development, and Cancer

    DEFF Research Database (Denmark)

    Laugesen, Anne; Helin, Kristian

    2014-01-01

    The chromatin environment is essential for the correct specification and preservation of cell identity through modulation and maintenance of transcription patterns. Many chromatin regulators are required for development, stem cell maintenance, and differentiation. Here, we review the roles...

  6. Chromatin damage induced by fast neutrons or UV laser radiation

    International Nuclear Information System (INIS)

    Radu, L.; Constantinescu, B.; Gazdaru, D.; Mihailescu, I.

    2002-01-01

    Chromatin samples from livers of Wistar rats were subjected to fast neutron irradiation in doses of 10-100 Gy or to a 248 nm excimer laser radiation, in doses of 0.5-3 MJ.m -2 . The action of the radiation on chromatin was monitored by chromatin intrinsic fluorescence and fluorescence lifetimes (of bound ethidium bromide to chromatin) and by analysing fluorescence resonance energy transfer between dansyl chloride and acridine orange coupled to chromatin. For the mentioned doses of UV excimer laser radiation, the action on chromatin was more intense than in the case of fast neutrons. The same types of damage are produced by the two radiations: acidic and basic destruction of chromatin protein structure, DNA strand breaking and the increase of the distance between DNA and proteins in chromatin. (author)

  7. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  8. Deoxyribonuclease probing of sea urchin embryo chromatin

    International Nuclear Information System (INIS)

    Landsman, D.

    1983-01-01

    The role that the sea urchin, Parechinus angulosus, embryo and sperm histone variants plays in chromatin structure has been investigated. Chromatin structure has been determined at different levels of resolution in sperm and in developing embryos using micrococcal nuclease, pancreatic deoxyribonuclease (DNase I) and restriction endonucleases. Micrococcal nuclease and restriction endonuclease digestions of sea urchin gastrula chromatin have been analysed and it is shown that it is not possible to isolate large polynucleosomal chromatin complexes which are soluble in low ionic strength buffers. The repeat length for sperm is significantly larger than blastula and gastrula repeat lengths whereas blastula and gastrula repeat lengths are not significantly different. Nucleosomal core particles have been isolated from early blastula, gastrula and sperm of sea urchins. After DNase I digestion of 5'-labelled core particles the rate constants of cutting of the DNA at the susceptible sites on these core particles have been determined. The DNase I digestion kinetics of blastula and gastrula core particles are similar whereas sperm core particles are digested at a slower rate, mainly at the sites which are closest to the ends of the core particle DNA

  9. Chromatin conformation capture strategies in molecular diagnostics

    NARCIS (Netherlands)

    de Vree, Pauline J.P.

    2015-01-01

    In this thesis I have explored the clinical potential of the 4C-technology and worked on development of a novel chromatin conformation capture based technology, called TLA. In chapter 2 I describe how the 4C-technology can be applied as a targeted strategy to identify putative fusion-genes or

  10. EBV Latency Types Adopt Alternative Chromatin Conformations

    Science.gov (United States)

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

    2011-01-01

    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

  11. EBV latency types adopt alternative chromatin conformations.

    Directory of Open Access Journals (Sweden)

    Italo Tempera

    2011-07-01

    Full Text Available 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.

  12. Chromatin organisation during Arabidopsis root development

    NARCIS (Netherlands)

    Lorvellec, M.

    2007-01-01

    The genetic information is stored in a highly compact manner in every nucleus. About 150 bp of DNA is packed around a histone octamer constituting a nucleosome. Nucleosomes are linked together by histone H1 and further compaction of this "beads on a string" form higher-order chromatin structures.

  13. Keystone Symposia on Epigenomics and Chromatin Dynamics

    DEFF Research Database (Denmark)

    Ravnskjær, Kim

    2012-01-01

    Keystone Symposia kicked off the start of 2012 with two joint meetings on Epigenomics and Chromatin Dynamics and a star-studded list of speakers. Held in Keystone, CO, January 17-22, and organized by Steven Jacobsen and Steven Henikoff and by Bradley Cairns and Geneviève Almouzni, respectively...

  14. The Latest Twists in Chromatin Remodeling.

    Science.gov (United States)

    Blossey, Ralf; Schiessel, Helmut

    2018-01-05

    In its most restrictive interpretation, the notion of chromatin remodeling refers to the action of chromatin-remodeling enzymes on nucleosomes with the aim of displacing and removing them from the chromatin fiber (the effective polymer formed by a DNA molecule and proteins). This local modification of the fiber structure can have consequences for the initiation and repression of the transcription process, and when the remodeling process spreads along the fiber, it also results in long-range effects essential for fiber condensation. There are three regulatory levels of relevance that can be distinguished for this process: the intrinsic sequence preference of the histone octamer, which rules the positioning of the nucleosome along the DNA, notably in relation to the genetic information coded in DNA; the recognition or selection of nucleosomal substrates by remodeling complexes; and, finally, the motor action on the nucleosome exerted by the chromatin remodeler. Recent work has been able to provide crucial insights at each of these three levels that add new twists to this exciting and unfinished story, which we highlight in this perspective. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell

    2013-01-01

    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...

  16. Chromatin organization and cellular sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Szumiel, I.; Walicka, M.

    1987-01-01

    The paper briefly describes chromatin organization in mammalian cells and reviews experimental work concerning relations between chromatin structure and accesibility of damaged DNA to repair enzymes. The ''contact effect'', the size of super-coiled DNA domains and ADP-ribosylation of chromatin proteins are discussed in relation to cellular radiosensitivity. 88 refs. (author)

  17. Chromatin Dynamics of the mouse β-globin locus

    NARCIS (Netherlands)

    M.P.C. van de Corput (Mariëtte); E. de Boer (Ernie); T.A. Knoch (Tobias); W.A. van Cappellen (Gert); M. Lesnussa (Michael); H.J.F.M.M. Eussen (Bert)

    2010-01-01

    textabstractLately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors

  18. Structural chromatin organization as a factor determining the rate of chromatin endonucleolysis in irradiated and intact thymocytes

    International Nuclear Information System (INIS)

    Ryabchenko, N.I.; Ivannik, B.P.

    1987-01-01

    A study was made of chromatin endonucleolysis in hypotonized thymocytes incubating in digestive buffers containing different concentrations of potassium, magnesium, calcium, and mercaptoethanol. Inhibition of endonucleolysis by univalent cation during the first 20 min of incubation was followed by intensive chromatin degradation. A decrease in free potassium content retarded chromatin degradation and enhanced the inhibiting effect of the univalent cations. The regularities of changes in the rate of chromatin endonucleolysis in different digestive buffers were similar with both exposed and intact thymocytes

  19. Pyrimidine dimers in Drosophila chromatin become increasingly accessible after irradiation

    International Nuclear Information System (INIS)

    Harris, P.V.; Boyd, J.B.

    1987-01-01

    A prokaryotic DNA-repair enzyme has been utilized as a probe for changes in the accessibility of pyrimidine dimers in Drosophila chromatin following UV irradiation. The results demonstrate a rapid cellular response to physiologically relevant doses of radiation which results in at least a 40% increase in accessible dimers. This increase occurs in two incision-deficient mutants which indicates that the excision-repair process, at or beyond the incision step, is not required or responsible for the increase. In the absence of excision the increase in accessibility persists for a least 2 days following irradiation. The observed increase in accessibility is inhibited by both novobiocin and coumermycin. These inhibitors do not inhibit the initial rate of incision, but do reduce dimer excision measured over more extended periods. A pre-incision process is proposed which actively exposes DNA lesions to excision repair. A fraction of the genome is postulated to be accessible without the intervention of that process. (Auth.)

  20. YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction

    DEFF Research Database (Denmark)

    Gabriele, Michele; Vulto-van Silfhout, Anneke T; Germain, Pierre-Luc

    2017-01-01

    that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin...... on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators....

  1. Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?

    Science.gov (United States)

    Fraser, L; Strzezek, J

    2007-07-15

    In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in

  2. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.; Jé gu, Teddy; Latrasse, David; Romero-Barrios, Natali; Christ, Auré lie; Benhamed, Moussa; Crespi, Martí n D.

    2014-01-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  3. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.

    2014-08-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  4. Chromatin Regulation of Neuronal Maturation and Plasticity.

    Science.gov (United States)

    Gallegos, David A; Chan, Urann; Chen, Liang-Fu; West, Anne E

    2018-05-01

    Neurons are dynamic cells that respond and adapt to stimuli throughout their long postmitotic lives. The structural and functional plasticity of neurons requires the regulated transcription of new gene products, and dysregulation of transcription in either the developing or adult brain impairs cognition. We discuss how mechanisms of chromatin regulation help to orchestrate the transcriptional programs that underlie the maturation of developing neurons and the plasticity of adult neurons. We review how chromatin regulation acts locally to modulate the expression of specific genes and more broadly to coordinate gene expression programs during transitions between cellular states. These data highlight the importance of epigenetic transcriptional mechanisms in postmitotic neurons. We suggest areas where emerging methods may advance understanding in the future. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. A transient ischemic environment induces reversible compaction of chromatin.

    Science.gov (United States)

    Kirmes, Ina; Szczurek, Aleksander; Prakash, Kirti; Charapitsa, Iryna; Heiser, Christina; Musheev, Michael; Schock, Florian; Fornalczyk, Karolina; Ma, Dongyu; Birk, Udo; Cremer, Christoph; Reid, George

    2015-11-05

    Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones. Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.

  6. Histone chaperone networks shaping chromatin function

    DEFF Research Database (Denmark)

    Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda

    2017-01-01

    and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....

  7. Probing chromatin structure with nuclease sensitivity assays.

    Science.gov (United States)

    Gregory, R I; Khosla, S; Feil, R

    2001-01-01

    To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

  8. [The biological aspects of chromatin diminution].

    Science.gov (United States)

    Akif'ev, A P; Grishanin, A K

    1993-01-01

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

  9. Default assembly of early adenovirus chromatin

    International Nuclear Information System (INIS)

    Spector, David J.

    2007-01-01

    In adenovirus particles, the viral nucleoprotein is organized into a highly compacted core structure. Upon delivery to the nucleus, the viral nucleoprotein is very likely to be remodeled to a form accessible to the transcription and replication machinery. Viral protein VII binds to intra-nuclear viral DNA, as do at least two cellular proteins, SET/TAF-Iβ and pp32, components of a chromatin assembly complex that is implicated in template remodeling. We showed previously that viral DNA-protein complexes released from infecting particles were sensitive to shearing after cross-linking with formaldehyde, presumably after transport of the genome into the nucleus. We report here the application of equilibrium-density gradient centrifugation to the analysis of the fate of these complexes. Most of the incoming protein VII was recovered in a form that was not cross-linked to viral DNA. This release of protein VII, as well as the binding of SET/TAF-Iβ and cellular transcription factors to the viral chromatin, did not require de novo viral gene expression. The distinct density profiles of viral DNA complexes containing protein VII, compared to those containing SET/TAF-Iβ or transcription factors, were consistent with the notion that the assembly of early viral chromatin requires both the association of SET/TAF-1β and the release of protein VII

  10. Capturing Structural Heterogeneity in Chromatin Fibers.

    Science.gov (United States)

    Ekundayo, Babatunde; Richmond, Timothy J; Schalch, Thomas

    2017-10-13

    Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    KAUST Repository

    Jé gu, Teddy; Domenichini, Sé verine; Blein, Thomas; Ariel, Federico; Christ, Auré lie; Kim, SoonKap; Crespi, Martin; Boutet-Mercey, Sté phanie; Mouille, Gré gory; Bourge, Mickaë l; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cé cile; 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.

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

    KAUST Repository

    Jégu, Teddy

    2015-10-12

    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.

  13. Spectroscopic study of fast-neutron-irradiated chromatin

    International Nuclear Information System (INIS)

    Radu, L.; Gazdaru, D.; Constantinescu, B.

    2004-01-01

    The effects produced by fast neutrons (0-100 Gy) on chromatin structure were analyzed by (i) [ 1 H]-NMR spectroscopy, (ii) time resolved spectroscopy, and (iii) fluorescence resonance energy transfer (FRET). Two types of chromatin were tested: (i) a chromatin from a normal tissue (liver of Wistar rats) and (ii) a chromatin from a tumoral tissue (Guerin limphotrope epithelioma, a rat solid tumor). The fast-neutron action on chromatin determines greater values of the [ 1 H]-NMR transverse relaxation time, indicating a more injured structure. Time-resolved fluorescence measurements show that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with increasing irradiation doses. This reflects the damage that occurs in DNA structure: production of single- and double-strand breaks due to sugar and base modifications. By the FRET method, the distance between dansyl chloride and acridine orange coupled at chromatin was determined. This distance increases upon fast-neutron action. The radiosensitivity of the tumor tissue chromatin seems higher than that of the normal tissue chromatin, probably because of its higher (loose) euchromatin/(compact) heterochromatin ratio. As the values of the physical parameters analyzed are specific for a determined dose, the establishment of these parameters may constitute a criterion for the microdosimetry of chromatin radiolesions produced by fast neutrons. (author)

  14. Spectroscopic study of fast-neutron-irradiated chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [V. Babes National Inst., Dept. of Molecular Genetics, Bucharest (Romania)]. E-mail: serbanradu@pcnet.ro; Gazdaru, D. [Bucharest Univ., Dept. of Biophysics, Physics Faculty, Bucharest (Romania); Constantinescu, B. [H. Hulubei National Inst., Dept. of Cyclotron, Bucharest (Romania)

    2004-02-01

    The effects produced by fast neutrons (0-100 Gy) on chromatin structure were analyzed by (i) [{sup 1}H]-NMR spectroscopy, (ii) time resolved spectroscopy, and (iii) fluorescence resonance energy transfer (FRET). Two types of chromatin were tested: (i) a chromatin from a normal tissue (liver of Wistar rats) and (ii) a chromatin from a tumoral tissue (Guerin limphotrope epithelioma, a rat solid tumor). The fast-neutron action on chromatin determines greater values of the [{sup 1}H]-NMR transverse relaxation time, indicating a more injured structure. Time-resolved fluorescence measurements show that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with increasing irradiation doses. This reflects the damage that occurs in DNA structure: production of single- and double-strand breaks due to sugar and base modifications. By the FRET method, the distance between dansyl chloride and acridine orange coupled at chromatin was determined. This distance increases upon fast-neutron action. The radiosensitivity of the tumor tissue chromatin seems higher than that of the normal tissue chromatin, probably because of its higher (loose) euchromatin/(compact) heterochromatin ratio. As the values of the physical parameters analyzed are specific for a determined dose, the establishment of these parameters may constitute a criterion for the microdosimetry of chromatin radiolesions produced by fast neutrons. (author)

  15. Chromosome aberration model combining radiation tracks, chromatin structure, DSB repair and chromatin mobility

    International Nuclear Information System (INIS)

    Friedland, W.; Kundrat, P.

    2015-01-01

    The module that simulates the kinetics and yields of radiation-induced chromosome aberrations within the biophysical code PARTRAC is described. Radiation track structures simulated by Monte Carlo methods are overlapped with multi-scale models of DNA and chromatin to assess the resulting DNA damage. Spatial mobility of individual DNA ends from double-strand breaks is modelled simultaneously with their processing by the non-homologous end-joining enzymes. To score diverse types of chromosome aberrations, the joined ends are classified regarding their original chromosomal location, orientation and the involvement of centromeres. A comparison with experimental data on dicentrics induced by gamma and alpha particles shows that their relative dose dependence is predicted correctly, although the absolute yields are overestimated. The critical model assumptions on chromatin mobility and on the initial damage recognition and chromatin remodelling steps and their future refinements to solve this issue are discussed. (authors)

  16. Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

    Directory of Open Access Journals (Sweden)

    Atsushi Fukuda

    2016-10-01

    Full Text Available In female mammals, activation of Xist (X-inactive specific transcript is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

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

    Science.gov (United States)

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

    2016-02-01

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

  18. Vacuum ultraviolet (VUV) absorption spectra of chromatin and its components

    International Nuclear Information System (INIS)

    Dodonova, N.Y.; Kiseleva, M.N.; Petrov, M.Y.; Tsyganenko, N.M.; Bubyakina, V.V.; Chikhirzhina, G.I.

    1984-01-01

    The electron absorption spectra of thin films of chromatin and chromatin components in the ultraviolet region (140-280 nm) were investigated. The absorption coefficients μ(lambda) of chromatin, nucleosomes with and without histone H1, total histones (TH), and DNA were compared. The spectra of nucleosomes differ from the sum-spectrum of DNA plus TH. The chromatin and nucleosome spectra are not similar in the spectral region of 190-160 nm. The lack of additivity of absorption coefficients at different wavelengths may be explained by different conformational changes of DNA, TH in nucleosomes and chromatin during the process of drying aqueous solutions for the preparation of thin films. The μ(lambda) values are useful for an estimate of the DNA and TH absorption in chromatin and nucleosomes in discussing UV and VUV irradiation damages. (Auth.)

  19. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Yolanda Stypula-Cyrus

    Full Text Available Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC. However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  20. Vibrational energy relaxation: proposed pathway of fast local chromatin denaturation

    International Nuclear Information System (INIS)

    Harder, D.; Greinert, R.

    2002-01-01

    The molecular mechanism responsible for the a component of exchange-type chromosome aberrations, of chromosome fragmentation and of reproductive cell death is one of the unsolved issues of radiation biology. Under review is whether vibrational energy relaxation in the constitutive biopolymers of chromatin, induced by inelastic energy deposition events and mediated via highly excited vibrational states, may provide a pathway of fast local chromatin denaturation, thereby producing the severe DNA lesion able to interact chemically with other, non-damaged chromatin. (author)

  1. Allosteric cross-talk in chromatin can mediate drug-drug synergy

    Science.gov (United States)

    Adhireksan, Zenita; Palermo, Giulia; Riedel, Tina; Ma, Zhujun; Muhammad, Reyhan; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2017-03-01

    Exploitation of drug-drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug-drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions.

  2. FACT prevents the accumulation of free histones evicted from transcribed chromatin and a subsequent cell cycle delay in G1.

    Directory of Open Access Journals (Sweden)

    Macarena Morillo-Huesca

    2010-05-01

    Full Text Available The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3 in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA-damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication.

  3. The Role of Chromatin-Associated Proteins in Cancer

    DEFF Research Database (Denmark)

    Helin, Kristian; Minucci, Saverio

    2017-01-01

    The organization of the chromatin structure is essential for maintaining cell-type-specific gene expression and therefore for cell identity. This structure is highly dynamic and is regulated by a large number of chromatin-associated proteins that are required for normal development...... and differentiation. Recurrent somatic mutations have been found with high frequency in genes coding for chromatin-associated proteins in cancer, and several of these are required for cancer maintenance. In this review, we discuss recent advances in understanding the role of chromatin-associated proteins...

  4. A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber

    DEFF Research Database (Denmark)

    Comet, Itys; Schuettengruber, Bernd; Sexton, Tom

    2011-01-01

    to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response...... element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3...... histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology....

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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: ► Stable knockdown of Brg1 and Brm enhances cellular sensitivity to cisplatin. ► Downregulation of Brg1 and Brm impedes the repair of cisplatin intrastrand adducts and interstrand crosslinks. ► Brg1 and Brm deficiency results in impaired chromatin relaxation, altered checkpoint activation as well as enhanced apoptosis. ► Downregulation of Brg1 and Brm affects recruitment of ERCC1, but not XPC to cisplatin DNA lesions.

  6. Histone modifications influence mediator interactions with chromatin

    Science.gov (United States)

    Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun; Liu, Zhongle; Quan, Amy; Costanzo, Michael; Dávila López, Marcela; Westholm, Jakub Orzechowski; Ronne, Hans; Boone, Charles; Gustafsson, Claes M.; Myers, Lawrence C.

    2011-01-01

    The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator—histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. PMID:21742760

  7. Homoeologous chromatin exchange in a radiation-induced gene transfer

    International Nuclear Information System (INIS)

    Dvorak, J.; Knott, D.R.

    1977-01-01

    Some of the ionizing-radiation-induced translocations between alien and wheat chromosomes show no deleterious effects and are transmitted normally through the pollen. Translocations of this type will be called ''compensating''. In one such compensating translocation, designated T4, it was found that chromatin in the long arm of wheat chromosome 7D was replaced with homoeologous chromatin of the Agropyron chromosome

  8. Homoeologous chromatin exchange in a radiation-induced gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    Dvorak, J; Knott, D R [Department of Crop Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    1977-03-01

    Some of the ionizing-radiation-induced translocations between alien and wheat chromosomes show no deleterious effects and are transmitted normally through the pollen. Translocations of this type will be called ''compensating''. In one such compensating translocation, designated T4, it was found that chromatin in the long arm of wheat chromosome 7D was replaced with homologous chromatin of the Agropyron chromosome.

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

    Science.gov (United States)

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

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

  10. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

    Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

  11. Nuclear visions enhanced: chromatin structure, organization and dynamics

    OpenAIRE

    Meshorer, Eran; Herrmann, Harald; Raška, Ivan

    2011-01-01

    The EMBO Workshop on ‘Chromatin Structure, Organization and Dynamics' took place in April 2011 in Prague, Czech Republic. Participants presented data on the generation of models of the genome, working to correlate changes in the organization of chromatin with the functional state of the genome.

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

    Science.gov (United States)

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

    2003-10-24

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

  13. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

  14. Fast neutron irradiation effects on liver chromatin structure

    International Nuclear Information System (INIS)

    Constantinescu, B.; Radu, L.

    1996-01-01

    The growing interest in neutron therapy requires complex studies on the mechanisms of neutron action on biological systems, especially on chromatin. The chromatin was extracted from a normal tissue-livers of Wistar rats - and from a tumoral tissue - Walker tumour maintained on Wistar rats. Irradiation doses from 5 Gy to 100 Gy by fast neutron intense beams produced via d(13.5 MeV) +Be (thick target) reaction at Bucharest U-120 Classical Cyclotron were used. To study the post-irradiation effects, various methods were employed. So, the variation in the 260 nm absorbency in chromatin thermal transition was pursuit. The chromatin-ethidium bromide complexes fluorescence with λ ex =480 nm and λ em =600 nm was analyzed. To determine chromatin DNA strand breaks a fluorimetric method, with cells' suspensions as starting material was used. This method requires a partial treatment with alkali producing three components: T-estimating the total fluorescence of DNA double helix, P-assigning the untwisting rate and B-the blank, where DNA is completely unfolded The percentsge of DNA double strand,-D-, remaining after this treatment, is: %D=100x(P-B)/(T-B). The intrinsic chromatin fluorescence was determined for tyrosine (λ ex =280 nm, λ em =305 nm), specific for badic chromatin prooteins, and for tryptophane (λ ex =290 nm, λ em =345 nm) specific for acid chromatin proteins. Polyacrylamide gel electrophoresis was performed: The double fluorescent labelling of chromatin was realized with acridine orange for DNA and with dansyl chloride for chromatin proteins. Fluorescence intensity determinations were done with λ ex =505 nm, λ em =530 nm for acridine orange and with λ ex =323 nm, λ em =505 nm for dansyl chloride. A Pye Unicam SP 1800 spectrophotometer and a Aminco SPF 500 spectrofluorimeter were employed. (author)

  15. Polymer physics predicts the effects of structural variants on chromatin architecture.

    Science.gov (United States)

    Bianco, Simona; Lupiáñez, Darío G; Chiariello, Andrea M; Annunziatella, Carlo; Kraft, Katerina; Schöpflin, Robert; Wittler, Lars; Andrey, Guillaume; Vingron, Martin; Pombo, Ana; Mundlos, Stefan; Nicodemi, Mario

    2018-05-01

    Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

  16. Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

    Science.gov (United States)

    Gácsi, Mariann; Antal, Otilia; Vasas, Gábor; Máthé, Csaba; Borbély, György; Saker, Martin L; Gyori, János; Farkas, Anna; Vehovszky, Agnes; Bánfalvi, Gáspár

    2009-06-01

    In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

  17. RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

    Science.gov (United States)

    Pascali, Chiara; Teichmann, Martin

    2013-01-01

    RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

  18. In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

    Science.gov (United States)

    Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

    2016-01-01

    Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

  19. Sedimentation Velocity Analysis of Large Oligomeric Chromatin Complexes Using Interference Detection.

    Science.gov (United States)

    Rogge, Ryan A; Hansen, Jeffrey C

    2015-01-01

    Sedimentation velocity experiments measure the transport of molecules in solution under centrifugal force. Here, we describe a method for monitoring the sedimentation of very large biological molecular assemblies using the interference optical systems of the analytical ultracentrifuge. The mass, partial-specific volume, and shape of macromolecules in solution affect their sedimentation rates as reflected in the sedimentation coefficient. The sedimentation coefficient is obtained by measuring the solute concentration as a function of radial distance during centrifugation. Monitoring the concentration can be accomplished using interference optics, absorbance optics, or the fluorescence detection system, each with inherent advantages. The interference optical system captures data much faster than these other optical systems, allowing for sedimentation velocity analysis of extremely large macromolecular complexes that sediment rapidly at very low rotor speeds. Supramolecular oligomeric complexes produced by self-association of 12-mer chromatin fibers are used to illustrate the advantages of the interference optics. Using interference optics, we show that chromatin fibers self-associate at physiological divalent salt concentrations to form structures that sediment between 10,000 and 350,000S. The method for characterizing chromatin oligomers described in this chapter will be generally useful for characterization of any biological structures that are too large to be studied by the absorbance optical system. © 2015 Elsevier Inc. All rights reserved.

  20. Chromatin decondensed by acetylation shows an elevated radiation response

    International Nuclear Information System (INIS)

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-01-01

    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

  1. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  2. Tracking the mechanical dynamics of human embryonic stem cell chromatin

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

  3. Anti-chromatin antibodies in juvenile rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    V. Gerloni

    2011-09-01

    Full Text Available Objective: to evaluate the prevalence and clinical significance of anti-chromatin antibodies (Abs in juvenile rheumatoid arthritis (JRA. Methods: IgG anti-chromatin Abs were detected by an enzyme-linked immunosorbent assay (ELISA, in sera of 94 children with JRA (10 children with systemic, 38 with polyarticular and 46 with oligoarticular disease onset. As control group, 33 age- and-sex-matched healthy children (HC were also examined. Results: Abs to chromatin were detected in 24/94 (25,5% of children suffering from JRA. Particularly, the higher prevalence of anti-chromatin Abs has been found in children with oligoarticular (30,4% and polyarticular (23,7% onset JRA. In these groups Abs titers were significantly higher compared to systemic JRA and HC (p=0.003. Anti-chromatin Abs were observed more frequently in patients with oligoarticular disease and chronic uveitis (21,7%. Furthermore, higher levels of anti-chromatin Abs has been found in all the patients treated with anti-TNFα therapy (p<0.0001. Conclusions: our results confirm previous data about the prevalence of anti-chromatin Abs in JRA. These Abs were significantly higher in the group of patients with oligoarticular onset with past or present hystory of ocular involvement and in the group with polyarticular JRA treated with biologic therapy. A long-term follow-up study could be useful to evaluate the potential utility of these autoantibodies.

  4. Massive nest-box supplementation boosts fecundity, survival and even immigration without altering mating and reproductive behaviour in a rapidly recovered bird population.

    Directory of Open Access Journals (Sweden)

    Karine Berthier

    Full Text Available Habitat restoration measures may result in artificially high breeding density, for instance when nest-boxes saturate the environment, which can negatively impact species' demography. Potential risks include changes in mating and reproductive behaviour such as increased extra-pair paternity, conspecific brood parasitism, and polygyny. Under particular cicumstances, these mechanisms may disrupt reproduction, with populations dragged into an extinction vortex. With the use of nuclear microsatellite markers, we investigated the occurrence of these potentially negative effects in a recovered population of a rare secondary cavity-nesting farmland bird of Central Europe, the hoopoe (Upupa epops. High intensity farming in the study area has resulted in a total eradication of cavity trees, depriving hoopoes from breeding sites. An intensive nest-box campaign rectified this problem, resulting in a spectacular population recovery within a few years only. There was some concern, however, that the new, high artificially-induced breeding density might alter hoopoe mating and reproductive behaviour. As the species underwent a serious demographic bottleneck in the 1970-1990s, we also used the microsatellite markers to reconstitute the demo-genetic history of the population, looking in particular for signs of genetic erosion. We found i a low occurrence of extra-pair paternity, polygyny and conspecific brood parasitism, ii a high level of neutral genetic diversity (mean number of alleles and expected heterozygosity per locus: 13.8 and 83%, respectively and, iii evidence for genetic connectivity through recent immigration of individuals from well differentiated populations. The recent increase in breeding density did thus not induce so far any noticeable detrimental changes in mating and reproductive behaviour. The demographic bottleneck undergone by the population in the 1970s-1990s was furthermore not accompanied by any significant drop in neutral genetic

  5. Massive nest-box supplementation boosts fecundity, survival and even immigration without altering mating and reproductive behaviour in a rapidly recovered bird population.

    Science.gov (United States)

    Berthier, Karine; Leippert, Fabio; Fumagalli, Luca; Arlettaz, Raphaël

    2012-01-01

    Habitat restoration measures may result in artificially high breeding density, for instance when nest-boxes saturate the environment, which can negatively impact species' demography. Potential risks include changes in mating and reproductive behaviour such as increased extra-pair paternity, conspecific brood parasitism, and polygyny. Under particular cicumstances, these mechanisms may disrupt reproduction, with populations dragged into an extinction vortex. With the use of nuclear microsatellite markers, we investigated the occurrence of these potentially negative effects in a recovered population of a rare secondary cavity-nesting farmland bird of Central Europe, the hoopoe (Upupa epops). High intensity farming in the study area has resulted in a total eradication of cavity trees, depriving hoopoes from breeding sites. An intensive nest-box campaign rectified this problem, resulting in a spectacular population recovery within a few years only. There was some concern, however, that the new, high artificially-induced breeding density might alter hoopoe mating and reproductive behaviour. As the species underwent a serious demographic bottleneck in the 1970-1990s, we also used the microsatellite markers to reconstitute the demo-genetic history of the population, looking in particular for signs of genetic erosion. We found i) a low occurrence of extra-pair paternity, polygyny and conspecific brood parasitism, ii) a high level of neutral genetic diversity (mean number of alleles and expected heterozygosity per locus: 13.8 and 83%, respectively) and, iii) evidence for genetic connectivity through recent immigration of individuals from well differentiated populations. The recent increase in breeding density did thus not induce so far any noticeable detrimental changes in mating and reproductive behaviour. The demographic bottleneck undergone by the population in the 1970s-1990s was furthermore not accompanied by any significant drop in neutral genetic diversity. Finally

  6. Proteomics of differential extraction fractions enriched for chromatin-binding proteins from colon adenoma and carcinoma tissues

    DEFF Research Database (Denmark)

    Knol, Jaco C; de Wit, Meike; Albrethsen, Jakob

    2014-01-01

    BACKGROUND: Altered nuclear and genomic structure and function are hallmarks of cancer cells. Research into nuclear proteins in human tissues could uncover novel molecular processes in cancer. Here, we examine biochemical tissue fractions containing chromatin-binding (CB) proteins in the context...... of colorectal cancer (CRC) progression. METHODS: CB protein-containing fractions were biochemically extracted from human colorectal tissues, including carcinomas with chromosomal instability (CIN), carcinomas with microsatellite instability (MIN), and adenomas. The CB proteins were subjected to label-free LC...

  7. Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1979-01-01

    Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA. (Auth.)

  8. Chromatin regulation at the frontier of synthetic biology

    Science.gov (United States)

    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

  9. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

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

    Science.gov (United States)

    Blum, Z; Lidin, S

    1988-08-01

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

  11. Mechanism of chromatin degradation in thymocytes of irradiated rats

    International Nuclear Information System (INIS)

    Zotova, R.N.; Umanskij, S.R.; Tokarskaya, V.I.

    1983-01-01

    A biphase change in poly (ADP-ribose) polymerase activity of the thymocyte chromatin was observed after 10 Gy irradiation of rats: during the first minutes the incorporation of 14 C-NAD increased by 40% then started decreasing to make 110, 60 and 35% after 1, 2 and 3 h, respectively. Irradiation of rat thymus chromatin in vitro sharply decreased poly (ADP-ribose) polymerase activity. The possible role of changes in the poly (ADP-ribose) synthesis in the activation of nuclear Ca/Mg-dependent endonuclease and in the postirradiation degradation of the thymocyte chromatin is discussed

  12. Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

    Directory of Open Access Journals (Sweden)

    Amy V Pointon

    2010-09-01

    Full Text Available Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Mice were treated with an acute dose of either doxorubicin (DOX (15 mg/kg or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ (25 mg/kg. DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO. Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these

  13. The SWI/SNF chromatin-remodeling gene AtCHR12 mediates temporary growth arrest in Arabidopsis thaliana upon perceiving environmental stress

    NARCIS (Netherlands)

    Mlynarova, L.; Nap, J.P.H.; Bisseling, T.

    2007-01-01

    One of the earliest responses of plants to environmental stress is establishing a temporary growth arrest that allows adaptation to adverse conditions. The response to abiotic stress requires the modulation of gene expression, which may be mediated by the alteration of chromatin structures. This

  14. Influence of chromatin molecular changes on RNA synthesis during embryonic development

    International Nuclear Information System (INIS)

    Chela-Flores, J.

    1990-12-01

    Two aspects of the chromatin repeat length (r l ) are discussed: (i) Why is r l longer for slowly dividing cells than in rapidly dividing cells?, and (ii) Why is the temporal evolution of r l a decreasing function of time (t) in mammalian cortical neurons whereas it is an increasing function of t for granule cells around the time of birth? These questions are discussed in terms of a hypothesis which assumes a correlation between deoxyribonucleic acid (DNA) packaging, transcription, and replication. (author). 27 refs

  15. Insights into Chromatin Structure and Dynamics in Plants

    Directory of Open Access Journals (Sweden)

    Stefanie Rosa

    2013-11-01

    Full Text Available 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.

  16. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang

    2016-02-04

    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.

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

    Science.gov (United States)

    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.

  18. histone H3 predominantly mark the pericentromeric chromatin

    Indian Academy of Sciences (India)

    SANTOSH KUMAR SHARMA

    pericentromeric chromatin during mitosis in monokinetic plants. J. Genet. .... bigger), cytological preparations (easy to difficult) as well as their habitat ... Poaceae. Monocot. Land. 14. Triticum aestivum. Common wheat. Poaceae. Monocot. Land.

  19. Neutron scattering studies on chromatin higher-order structure

    Energy Technology Data Exchange (ETDEWEB)

    Graziano, V.; Gerchman, S.E.; Schneider, D.K.; Ramakrishnan, V. [Brookhaven National Laboratory, Upton, NY (United States)

    1994-12-31

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

  20. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin

    Science.gov (United States)

    Bandaria, Jigar N.; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet

    2016-01-01

    SUMMARY 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, and 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 become more accessible to telomere-associated proteins and accumulate DDR signals. 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

  1. Determination of local chromatin composition by CasID.

    Science.gov (United States)

    Schmidtmann, Elisabeth; Anton, Tobias; Rombaut, Pascaline; Herzog, Franz; Leonhardt, Heinrich

    2016-09-02

    Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition.

  2. Aberrant Chromatin Modification as a Mechanism of Prostate Cancer Progression

    National Research Council Canada - National Science Library

    Chen, Hongwu

    2004-01-01

    .... However, the underlying mechanism is still unclear. The purpose of this study is to test the hypothesis that aberrant chromatin modification plays a critical role in prostate cancer progression...

  3. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang; Schwarzer, Dirk

    2016-01-01

    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

  4. FACT facilitates chromatin transcription by RNA polymerases I and III

    DEFF Research Database (Denmark)

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...... in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III...

  5. Neutron scattering studies on chromatin higher-order structure

    International Nuclear Information System (INIS)

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

    1994-01-01

    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

  6. Citrullination regulates pluripotency and histone H1 binding to chromatin

    DEFF Research Database (Denmark)

    Christophorou, Maria A; Castelo-Branco, Gonçalo; Halley-Stott, Richard P

    2014-01-01

    citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune...... and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel...... PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic...

  7. histone H3 predominantly mark the pericentromeric chromatin

    Indian Academy of Sciences (India)

    SANTOSH KUMAR SHARMA

    packaging of eukaryotic DNA in nucleoprotein complex known as .... The plant material used in the present study has ... materials (root tips/flower buds) were fixed in PHEMES ..... fications that mark active chromatin, while there are no data.

  8. Cytogenetic abnormality in man, wider implications of theories of sex chromatin origin.

    Science.gov (United States)

    MILES, C P

    1962-01-01

    Female nuclei may be identified by means of sex chromatin. In general the number of sex chromatin bodies is one less than the number of X chromosomes. An exception to this rule is a case of sex chromatin-positive XO Turner's syndrome. This case suggests the possibility of sex chromatin-positive XY males, and it may be evidence for chromosomal differentiation.

  9. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    OpenAIRE

    Christopher Ian Cazzonelli; Nazia eNisar; Andrea C Roberts; Kevin eMurray; Justin O Borevitz; Barry James Pogson

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzy...

  10. A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

    OpenAIRE

    Cazzonelli, Christopher I.; Nisar, Nazia; Roberts, Andrea C.; Murray, Kevin D.; Borevitz, Justin O.; Pogson, Barry J.

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzym...

  11. Mechanism of chromatin degradation in thymocytes of irradiated rats

    International Nuclear Information System (INIS)

    Nikonova, L.V.; Nelipovich, P.A.; Umanskij, S.R.

    1983-01-01

    Chromatin digestion in isolated thymocyte nuclei with DNAase I, micrococcal nuclease and nuclease from Serratia marcescens was studied. It was shown that 3 h after irradiation (10 Gy), the kinetics of accumulation of acid soluble and salt soluble products of DNA degradation, caused by exogenous nucleases, remains unchanged. The administration of cycloheximide does not influence the sensitivity of chromatin to DNAase I and somewhat increases the rate of salt soluble products formation upon the nuclease from S, marcescens treatment

  12. ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

    Directory of Open Access Journals (Sweden)

    Alexandra Lusser

    2011-10-01

    Full Text Available ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

  13. Fragmentation of chromatin with 125I radioactive disintegrations

    International Nuclear Information System (INIS)

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

    1976-01-01

    The DNA in Chinese hamster cells was labeled first for 3 h with [ 3 H]TdR and then for 3 h with [ 125 I]UdR. Chromatin was extracted, frozen, and stored at -30 0 C until 1.0 x 10 17 and 1.25 x 10 17 disintegrations/g of labeled DNA occurred for 125 I and 3 H, respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125 I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [ 125 I] chromatin into pieces smaller than the [ 3 H] chromatin. In other words, 125 I disintegrations caused much more localized damage in the chromatin labeled with 125 I than in the chromatin labeled with 3 H, and fragments induced in DNA by 125 I disintegrations were not held together by the associated chromosomal proteins. Use of this 125 I 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

  14. Cytoplasmic chromatin triggers inflammation in senescence and cancer.

    Science.gov (United States)

    Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

    2017-10-19

    Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

  15. Chromatin modifications and the DNA damage response to ionizing radiation

    International Nuclear Information System (INIS)

    Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

  16. Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research.

    Science.gov (United States)

    Howe, Elizabeth S; Clemente, Thomas E; Bass, Hank W

    2012-06-01

    Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

  17. TIP48/Reptin and H2A.Z requirement for initiating chromatin remodeling in estrogen-activated transcription.

    Directory of Open Access Journals (Sweden)

    Mathieu Dalvai

    2013-04-01

    Full Text Available Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. The role of H2A.Z at these sites remains poorly characterized. Our study investigates changes in the chromatin environment at the Cyclin D1 gene (CCND1 during transcriptional initiation in response to estradiol in estrogen receptor positive mammary tumour cells. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of CCND1 when the gene is poorly transcribed. Stimulation of CCND1 expression required release of H2A.Z concomitantly from both these DNA elements. The AAA+ family members TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin environment at CCND1 as a prerequisite for binding of the estrogen receptor (ERα in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z, which triggers a dissociation of the CCND1 3' enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables the estrogen receptor to bind to the CCND1 promoter. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Dynamic release of gene loops could be a rapid means to remodel chromatin and to stimulate transcription in response to hormones.

  18. Circadian cycle-dependent MeCP2 and brain chromatin changes.

    Directory of Open Access Journals (Sweden)

    Alexia Martínez de Paz

    Full Text Available Methyl CpG binding protein 2 (MeCP2 is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.

  19. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracks.

    Science.gov (United States)

    Bacher, Christian P; Reichenzeller, Michaela; Athale, Chaitanya; Herrmann, Harald; Eils, Roland

    2004-11-23

    . We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

  20. The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

    Science.gov (United States)

    Xue, Yutong; Gibbons, Richard; Yan, Zhijiang; Yang, Dafeng; McDowell, Tarra L; Sechi, Salvatore; Qin, Jun; Zhou, Sharleen; Higgs, Doug; Wang, Weidong

    2003-09-16

    ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

  1. Cumulative Impact of Polychlorinated Biphenyl and Large Chromosomal Duplications on DNA Methylation, Chromatin, and Expression of Autism Candidate Genes.

    Science.gov (United States)

    Dunaway, Keith W; Islam, M Saharul; Coulson, Rochelle L; Lopez, S Jesse; Vogel Ciernia, Annie; Chu, Roy G; Yasui, Dag H; Pessah, Isaac N; Lott, Paul; Mordaunt, Charles; Meguro-Horike, Makiko; Horike, Shin-Ichi; Korf, Ian; LaSalle, Janine M

    2016-12-13

    Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Molecular and Biochemical Methods Useful for the Epigenetic Characterization of Chromatin-Associated Proteins in Bivalve Molluscs

    Directory of Open Access Journals (Sweden)

    Ciro Rivera-Casas

    2017-08-01

    Full Text Available Bivalve molluscs constitute a ubiquitous taxonomic group playing key functions in virtually all ecosystems, and encompassing critical commercial relevance. Along with a sessile and filter-feeding lifestyle in most cases, these characteristics make bivalves model sentinel organisms routinely used for environmental monitoring studies in aquatic habitats. The study of epigenetic mechanisms linking environmental exposure and specific physiological responses (i.e., environmental epigenetics stands out as a very innovative monitoring strategy, given the role of epigenetic modifications in acclimatization and adaptation. Furthermore, the heritable nature of many of those modifications constitutes a very promising avenue to explore the applicability of epigenetic conditioning and selection in management and restoration strategies. Chromatin provides a framework for the study of environmental epigenetic responses. Unfortunately, chromatin and epigenetic information are very limited in most non-traditional model organisms and even completely lacking in most environmentally and ecologically relevant organisms. The present work aims to provide a comprehensive and reproducible experimental workflow for the study of bivalve chromatin. First, a series of guidelines for the molecular isolation of genes encoding chromatin-associated proteins is provided, including information on primers suitable for conventional PCR, Rapid Amplification of cDNA Ends (RACE, genome walking and quantitative PCR (qPCR experiments. This section is followed by the description of methods specifically developed for the analysis of histone and SNBP proteins in different bivalve tissues, including protein extraction, purification, separation and immunodetection. Lastly, information about available antibodies, their specificity and performance is also provided. The tools and protocols described here complement current epigenetic analyses (usually limited to DNA methylation by incorporating

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

    International Nuclear Information System (INIS)

    Terry, Samantha Y.A.; Vallis, Katherine A.

    2012-01-01

    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 (γH2AX assay) and clonogenic survival were evaluated after exposure to 111 In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of 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 γH2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 μM) compared with IR alone (16 ± 0.6 and 14 ± 0.3 vs. 12 ± 0.4 and 11 ± 0.2, respectively). More γH2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to 111 In-DTPA-hEGF (6 MBq/μg) plus SAHA vs. 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 111 In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 μM) vs. IR alone (0.6% ± 0.01 and 0.3% ± 0.2 vs. 5.8% ± 0.2 and 2% ± 0.1, respectively) and after 111 In-DTPA-hEGF plus SAHA compared to 111 In-DTPA-hEGF alone (21% ± 0.4% and 19% ± 4.6 vs. 33% ± 2.3 and 32% ± 3.7). SAHA did not affect 111 In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer γH2AX foci per cell after IR and 111 In-DTPA-hEGF compared to controls but did not significantly alter clonogenic

  4. Circadian expression profiles of chromatin remodeling factor genes in Arabidopsis.

    Science.gov (United States)

    Lee, Hong Gil; Lee, Kyounghee; Jang, Kiyoung; Seo, Pil Joon

    2015-01-01

    The circadian clock is a biological time keeper mechanism that regulates biological rhythms to a period of approximately 24 h. The circadian clock enables organisms to anticipate environmental cycles and coordinates internal cellular physiology with external environmental cues. In plants, correct matching of the clock with the environment confers fitness advantages to plant survival and reproduction. Therefore, circadian clock components are regulated at multiple layers to fine-tune the circadian oscillation. Epigenetic regulation provides an additional layer of circadian control. However, little is known about which chromatin remodeling factors are responsible for circadian control. In this work, we analyzed circadian expression of 109 chromatin remodeling factor genes and identified 17 genes that display circadian oscillation. In addition, we also found that a candidate interacts with a core clock component, supporting that clock activity is regulated in part by chromatin modification. As an initial attempt to elucidate the relationship between chromatin modification and circadian oscillation, we identified novel regulatory candidates that provide a platform for future investigations of chromatin regulation of the circadian clock.

  5. Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population.

    Science.gov (United States)

    Singh, Virendra; Singh, Laishram C; Singh, Avninder P; Sharma, Jagannath; Borthakur, Bibhuti B; Debnath, Arundhati; Rai, Avdhesh K; Phukan, Rup K; Mahanta, Jagadish; Kataki, Amal C; Kapur, Sujala; Saxena, Sunita

    2015-01-01

    Esophageal cancer incidence is reported in high frequency in northeast India. The etiology is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco/betel quid chewing and alcohol habits. Since DNA methylation, histone modification and miRNA-mediated epigenetic processes alter the gene expression, the involvement of these processes might be useful to find out epigenetic markers of esophageal cancer risk in northeast Indian population. The present investigation was aimed to carryout differential expression profiling of chromatin modification enzymes in tumor and normal tissue collected from esophageal squamous cell carcinoma (ESCC) patients. Differential mRNA expression profiling and their validation was done by quantitative real time PCR and tissue microarray respectively. Univariate and multiple logistic regression analysis were used to analyze the epidemiological data. mRNA expression data was analyzed by Student t-test. Fisher exact test was used for tissue microarray data analysis. Higher expression of enzymes regulating methylation (DOT1L and PRMT1) and acetylation (KAT7, KAT8, KAT2A and KAT6A) of histone was found associated with ESCC risk. Tissue microarray done in independent cohort of 75 patients revealed higher nuclear protein expression of KAT8 and PRMT1 in tumor similar to mRNA expression. Expression status of PRMT1 and KAT8 was found declined as we move from low grade to high grade tumor. Betel nut chewing, alcohol drinking and dried fish intake were significantly associated with increased risk of esophageal cancer among the study subject. Study suggests the association of PRMT1 and KAT8 with esophageal cancer risk and its involvement in the transition process of low to high grade tumor formation. The study exposes the differential status of chromatin modification enzymes between tumor and normal tissue and points out that relaxed state of chromatin facilitates more transcriptionally active

  6. Higher-order structure of Saccharomyces cerevisiae chromatin

    International Nuclear Information System (INIS)

    Lowary, P.T.; Widom, J.

    1989-01-01

    We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast killer virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure

  7. Chromatin Regulation and the Histone Code in HIV Latency
.

    Science.gov (United States)

    Turner, Anne-Marie W; Margolis, David M

    2017-06-01

    The formation of a latent reservoir of Human Immunodeficiency Virus (HIV) infection hidden from immune clearance remains a significant obstacle to approaches to eradicate HIV infection. Towards an understanding of the mechanisms of HIV persistence, there is a growing body of work implicating epigenetic regulation of chromatin in establishment and maintenance of this latent reservoir. Here we discuss recent advances in the field of chromatin regulation, specifically in our understanding of the histone code, and how these discoveries relate to our current knowledge of the chromatin mechanisms linked to HIV transcriptional repression and the reversal of latency. We also examine mechanisms unexplored in the context of HIV latency and briefly discuss current therapies aimed at the induction of proviral expression within latently infected cells. We aim to emphasize that a greater understanding of the epigenetic mechanisms which govern HIV latency could lead to new therapeutic targets for latency reversal and clearance cure strategies.

  8. Fast neutron biological effects on normal and tumor chromatin

    International Nuclear Information System (INIS)

    Constantinescu, B.; Bugoi, Roxana; Paunica, Tatiana; Radu, Liliana

    1997-01-01

    Growing interest in neutron therapy and radioprotection requires complex studies on the mechanisms of neutron action on biological systems, especially on chromatin (the complex of deoxyribonucleic acid-DNA- with proteins in eukaryotic cells). Our study aims to investigate the fast neutrons induced damages in normal and tumor chromatin, studying thermal transition, intrinsic fluorescence and fluorescence of chromatin-ethidium bromide complexes behavior versus irradiation dose. The Bucharest U-120 variable energy Cyclotron was employed as an intense source of fast neutrons produced by 13.5 MeV deuterons on a thick beryllium target (166.5 mg/cm 2 ) placed at 20 angle against the incident beam. The average energy is 5.24 MeV. The total yield at 0 angle is 6.7 x 10 16 n/sr·C·MeV. To determine neutron and gamma irradiation doses, home made thermoluminescent detectors-TLD(γ) and TLD (γ + n) were used: for gamma MgF 2 : Mn mixed with Teflon pellets (φ 12.5 mm, 0.6±0.1 mm thick) and for gamma plus neutrons MgF 2 :Mn mixed with 6 LiF and Teflon pellets (same dimensions). Using a 8.022 x 10 -2 albedo factor value and the equivalence 1Gy (n)=2·10 10 fast neutron/cm 2 , the dose for the irradiation of 1.2 x 10 2 Gy/μC, with an estimated precision of 15% C for neutrons and 7.8 x 10 -4 Gy/μC for gamma, at 10 cm behind Be target, was found, respectively. A diminution of the negative fluorescence intensity for chromatin-ethidium bromide complexes with the increasing of neutron dose (from 0.98 at 5 Gy to 0.85 at 100 Gy) was observed for normal chromatin. This fact reflects chromatin DNA injuries, with the decrease of double helix DNA proportion. To study the influence of gyrostan, thyroxine and D3 vitamin treatments on fast neutron radiolysis in tumor chromatin,10 mg/kg of anticancer drug gyrostan, 40μg/kg of hormonal compound thyroxine and 30,000 IU/kg of D3 vitamin were administrated, separately or associated, to Wistar rats bearing Walker carcinosarcoma. Representing

  9. Radiation-induced cell death by chromatin loss

    International Nuclear Information System (INIS)

    Campbell, I.R.; Warenius, H.M.

    1989-01-01

    A model is proposed which relates reproductive death of cells caused by radiation to loss of chromatin at cell division. This loss of chromatin can occur through chromosomal deletions or through the formation of asymmetrical chromosomal exchanges. It is proposed that smaller doses of radiation produce fewer chromatin breaks, which are more likely to be accurately repaired, compared with larger doses. Consequently, smaller doses of radiation are less efficient in causing cell death, leading to a shoulder on the cell survival curve. Experimental evidence supports this model, and the fit between the derived formula and experimental cell survival curves is good. The derived formula approximates to the linear-quadratic equation at low doses of radiation. (author)

  10. INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division

    Directory of Open Access Journals (Sweden)

    Graeme J. Gowans

    2018-01-01

    Full Text Available Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the yeast metabolic cycle (YMC. Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here, we demonstrate that the INO80 chromatin-remodeling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC1-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin to restrict proliferation to metabolically optimal states.

  11. ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

    Science.gov (United States)

    Horard, Béatrice; Sapey-Triomphe, Laure; Bonnefoy, Emilie; Loppin, Benjamin

    2018-05-11

    Anti-Silencing Factor 1 (ASF1) is a conserved H3-H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosome assembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3-H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatin assembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. Here, we have investigated the implication of ASF1 in these two distinct assembly processes. We show that depletion of the maternal pool of ASF1 with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, we show that asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, we found that ASF1 is required for the formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, we show that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility. We conclude that ASF1 is critically required to load H3.3-H4 dimers on the HIRA complex prior to histone deposition on paternal DNA. This separation of tasks could optimize the rapid assembly of paternal chromatin within the gigantic volume of the egg cell. In contrast, ASF1 is surprisingly dispensable for the

  12. Chromatin structure and evolution in the human genome

    Directory of Open Access Journals (Sweden)

    Dunlop Malcolm G

    2007-05-01

    Full Text Available Abstract Background Evolutionary rates are not constant across the human genome but genes in close proximity have been shown to experience similar levels of divergence and selection. The higher-order organisation of chromosomes has often been invoked to explain such phenomena but previously there has been insufficient data on chromosome structure to investigate this rigorously. Using the results of a recent genome-wide analysis of open and closed human chromatin structures we have investigated the global association between divergence, selection and chromatin structure for the first time. Results In this study we have shown that, paradoxically, synonymous site divergence (dS at non-CpG sites is highest in regions of open chromatin, primarily as a result of an increased number of transitions, while the rates of other traditional measures of mutation (intergenic, intronic and ancient repeat divergence as well as SNP density are highest in closed regions of the genome. Analysis of human-chimpanzee divergence across intron-exon boundaries indicates that although genes in relatively open chromatin generally display little selection at their synonymous sites, those in closed regions show markedly lower divergence at their fourfold degenerate sites than in neighbouring introns and intergenic regions. Exclusion of known Exonic Splice Enhancer hexamers has little affect on the divergence observed at fourfold degenerate sites across chromatin categories; however, we show that closed chromatin is enriched with certain classes of ncRNA genes whose RNA secondary structure may be particularly important. Conclusion We conclude that, overall, non-CpG mutation rates are lowest in open regions of the genome and that regions of the genome with a closed chromatin structure have the highest background mutation rate. This might reflect lower rates of DNA damage or enhanced DNA repair processes in regions of open chromatin. Our results also indicate that dS is a poor

  13. Model for the structure of the active nucleolar chromatin

    International Nuclear Information System (INIS)

    Labhart, P.; Ness, P.; Banz, E.; Parish, R.; Koller, T.; Universitaet Zurich, Switzerland)

    1983-01-01

    Transcribed ribosomal genes of Xenopus laevis oocytes and of Dictyostelium discoideum were studied electron microscopically using step gradients at different ionic strengths. Under these conditions the fiber of the active chromatin appears smooth and is indistinguishable from free DNA. The accessibility of the coding region and of a nontranscribed spacer region to restriction enzymes and micrococcal nuclease were investigated. All of the results obtained are consistent with a model in which active nucleolar chromatin is mostly composed of free DNA and the components required for transcription. 50 references, 7 figures

  14. Chromatin Structure of Epstein-Barr Virus Latent Episomes.

    Science.gov (United States)

    Lieberman, Paul M

    2015-01-01

    EBV latent infection is characterized by a highly restricted pattern of viral gene expression. EBV can establish latent infections in multiple different tissue types with remarkable variation and plasticity in viral transcription and replication. During latency, the viral genome persists as a multi-copy episome, a non-integrated-closed circular DNA with nucleosome structure similar to cellular chromosomes. Chromatin assembly and histone modifications contribute to the regulation of viral gene expression, DNA replication, and episome persistence during latency. This review focuses on how EBV latency is regulated by chromatin and its associated processes.

  15. A Method to Identify Nucleolus-Associated Chromatin Domains (NADs).

    Science.gov (United States)

    Carpentier, Marie-Christine; Picart-Picolo, Ariadna; Pontvianne, Frédéric

    2018-01-01

    The nuclear context needs to be taken into consideration to better understand the mechanisms shaping the epigenome and its organization, and therefore its impact on gene expression. For example, in Arabidopsis, heterochromatin is preferentially localized at the nuclear and the nucleolar periphery. Although chromatin domains associating with the nuclear periphery remain to be identified in plant cells, Nucleolus Associated chromatin Domains (NADs) can be identified thanks to a protocol allowing the isolation of pure nucleoli. We describe here the protocol enabling the identification of NADs in Arabidopsis. Providing the transfer of a nucleolus marker as described here in other crop species, this protocol is broadly applicable.

  16. The importance of topoisomerases for chromatin regulated genes

    DEFF Research Database (Denmark)

    Fredsøe, Jacob Christian; Pedersen, Jakob Madsen; Rødgaard, Morten Terpager

    2013-01-01

    DNA topoisomerases are enzymes, which function to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the activation of chromatin...... topoisomerases for optimal activation, but in contrast to the PHO5 gene, topoisomerases are not required for chromatin remodeling of the GAL1/10 promoter region, indicating a different role of the enzymes. We are currently performing a detailed investigation of the GAL genes to elucidate the precise role...

  17. Retention of the Native Epigenome in Purified Mammalian Chromatin.

    Directory of Open Access Journals (Sweden)

    Andreas H Ehrensberger

    Full Text Available A protocol is presented for the isolation of native mammalian chromatin as fibers of 25-250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.

  18. FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription

    International Nuclear Information System (INIS)

    Holmqvist, Per-Henrik; Belikov, Sergey; Zaret, Kenneth S.; Wrange, Oerjan

    2005-01-01

    Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription. The proximal FoxA binding segment consisted of two individual FoxA sites at -57/-46 and -45/-34, respectively, that mediated an increased basal MMTV transcription. FoxA1 binding altered the chromatin structure of both the inactive- and the hormone-activated MMTV LTR. Hydroxyl radical foot printing revealed FoxA1-mediated changes in the nucleosome arrangement. Micrococcal nuclease digestion showed the hormone-dependent sub-nucleosome complex, containing ∼120 bp of DNA, to be expanded by FoxA1 binding to the proximal segment into a larger complex containing ∼200 bp. The potential function of the FoxA1-mediated expression of the MMTV provirus for maintenance of expression in different tissues is discussed

  19. Dancing on damaged chromatin. Functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage

    International Nuclear Information System (INIS)

    Iijima, Kenta; Ohara, Maki; Seki, Ryota; Tauchi, Hiroshi

    2008-01-01

    In order to preserve and protect genetic information, eukaryotic cells have developed a signaling or communications network to help the cell respond to DNA damage, and ATM and NBS1 are key players in this network. ATM is a protein kinase which is activated immediately after a DNA double strand break (DSB) is formed, and the resulting signal cascade generated in response to cellular DSBs is regulated by post-translational protein modifications such as phosphorylation and acetylation. In addition, to ensure the efficient functioning of DNA repair and cell cycle checkpoints, the highly ordered structure of eukaryotic chromatin must be appropriately altered to permit access of repair-related factors to DNA. These alterations are termed chromatin remodeling, and are executed by a specific remodeling complex in conjunction with histone modifications. Current advances in the molecular analysis of DNA damage responses have shown that the auto-phosphorylation of ATM and the interaction between ATM and NBS1 are key steps for ATM activation, and that the association of ATM and NBS1 is involved in chromatin remodeling. Identification of novel factors which function in ubiquitination (RNF8, Ubc13, Rap80, etc.) has also enabled us to understand more details of the early stages in DNA repair pathways which respond to DSBs. In this review, the focus is on the role of ATM and the RAD50/MRE11/NBS1 complex in DSB response pathways, and their role in DSB repair and in the regulation of chromatin remodeling. (author)

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

    Science.gov (United States)

    Savic, Velibor

    2013-01-01

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

  1. Large-scale Comparative Study of Hi-C-based Chromatin 3D Structure Modeling Methods

    KAUST Repository

    Wang, Cheng

    2018-01-01

    Chromatin is a complex polymer molecule in eukaryotic cells, primarily consisting of DNA and histones. Many works have shown that the 3D folding of chromatin structure plays an important role in DNA expression. The recently proposed Chro- mosome

  2. Testing Whether Defective Chromatin Assembly in S-Phase Contributes to Breast Cancer

    National Research Council Canada - National Science Library

    Adams, Peter

    2003-01-01

    .... We used a dominant negative mutant of (chromatin assembly factor-I) CAF1, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S-phase chromatin assembly and found that this induced S-phase arrest...

  3. Testing Whether Defective Chromatin Assembly in S-Phase Contributes to Breast Cancer

    National Research Council Canada - National Science Library

    Adams, Peter

    2004-01-01

    .... We used a dominant negative mutant of (chromatin assembly factor-I) CAF1, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S-phase chromatin assembly and found that this induced S-phase arrest...

  4. Relationship between chromatin structure and sensitivity to molecularly targeted auger electron radiation therapy.

    NARCIS (Netherlands)

    Terry, S.Y.A.; Vallis, K.A.

    2012-01-01

    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

  5. Chromatin-bound MDM2, a new player in metabolism.

    Science.gov (United States)

    Riscal, Romain; Le Cam, Laurent; Linares, Laetitia K

    2016-01-01

    The oncoprotein MDM2 is recognized as a major negative regulator of the p53 tumor suppressor but growing evidence indicates that its oncogenic activities extend beyond p53. We show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis.

  6. SAGA, TFIID and regulation of transcription through chromatin

    NARCIS (Netherlands)

    Schram, A.W.

    2013-01-01

    Chromatin has an important role in eukaryotic transcription. Research into this role is ongoing and genome-wide analysis has correlated various histone modifications to multiple elements in active and silent genes, such as enhancers, promoters and coding regions. Modifications often serve to recruit

  7. Interaction of maize chromatin-associated HMG proteins with mononucleosomes

    DEFF Research Database (Denmark)

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...

  8. Role of chromatin factors in Arabidopsis root stem cell maintenance

    NARCIS (Netherlands)

    Kornet, N.G.

    2008-01-01

    Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique

  9. Control of trichome branching by Chromatin Assembly Factor-1

    Directory of Open Access Journals (Sweden)

    Hennig Lars

    2008-05-01

    Full Text Available Abstract Background Chromatin dynamics and stability are both required to control normal development of multicellular organisms. Chromatin assembly factor CAF-1 is a histone chaperone that facilitates chromatin formation and the maintenance of specific chromatin states. In plants and animals CAF-1 is essential for normal development, but it is poorly understood which developmental pathways require CAF-1 function. Results Mutations in all three CAF-1 subunits affect Arabidopsis trichome morphology and lack of CAF-1 function results in formation of trichomes with supernumerary branches. This phenotype can be partially alleviated by external sucrose. In contrast, other aspects of the CAF-1 mutant phenotype, such as defective meristem function and organ formation, are aggravated by external sucrose. Double mutant analyses revealed epistatic interactions between CAF-1 mutants and stichel, but non-epistatic interactions between CAF-1 mutants and glabra3 and kaktus. In addition, mutations in CAF-1 could partly suppress the strong overbranching and polyploidization phenotype of kaktus mutants. Conclusion CAF-1 is required for cell differentiation and regulates trichome development together with STICHEL in an endoreduplication-independent pathway. This function of CAF-1 can be partially substituted by application of exogenous sucrose. Finally, CAF-1 is also needed for the high degree of endoreduplication in kaktus mutants and thus for the realization of kaktus' extreme overbranching phenotype.

  10. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

  11. Epigenetic regulation and chromatin remodeling in learning and memory.

    Science.gov (United States)

    Kim, Somi; Kaang, Bong-Kiun

    2017-01-13

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

  12. Effect of triiodothyronine on rat liver chromatin protein kinase

    International Nuclear Information System (INIS)

    Kruh, J.; Tichonicky, L.

    1976-01-01

    1) Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2) Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32 P when incubated with [γ- 32 P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32 P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins. (orig.) [de

  13. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-01-01

    to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results

  14. Chromatin-regulating proteins as targets for cancer therapy

    International Nuclear Information System (INIS)

    Oike, Takahiro; Ogiwara, Hideaki; Kohno, Takashi; Amornwichet, Napapat; Nakano, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. (author)

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

    Science.gov (United States)

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

    2016-02-01

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

  16. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

    DEFF Research Database (Denmark)

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin

    2014-01-01

    , histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin- associated proteins and identified a Haspin...

  17. Chromatin Structure in Cell Differentiation, Aging and Cancer

    NARCIS (Netherlands)

    S. Kheradmand Kia (Sima)

    2009-01-01

    textabstractChromatin is the structure that the eukaryotic genome is packaged into, allowing over a metre of DNA to fit into the small volume of the nucleus. It is composed of DNA and proteins, most of which are histones. This DNA-protein complex is the template for a number of essential cell

  18. A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation.

    Science.gov (United States)

    Nohara, Kazunari; Chen, Zheng; Yoo, Seung-Hee

    2017-07-06

    Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

  19. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators.

    Directory of Open Access Journals (Sweden)

    So Yeon Kwon

    2016-04-01

    Full Text Available NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.

  20. Myocardial thallium-201 kinetics and regional flow alterations with 3 hours of coronary occlusion and either rapid reperfusion through a totally patent vessel or slow reperfusion through a critical stenosis

    International Nuclear Information System (INIS)

    Granato, J.E.; Watson, D.D.; Flanagan, T.L.; Beller, G.A.

    1987-01-01

    Myocardial thallium-201 kinetics and regional blood flow alterations were examined in a canine model using 3 hours of coronary occlusion and different methods of reperfusion. Group I comprised 10 dogs undergoing a 3 hour left anterior descending artery occlusion and no reperfusion. Group II comprised seven dogs undergoing 3 hours of left anterior descending artery occlusion and rapid reperfusion through a totally patent vessel. Group III comprised 10 dogs undergoing 3 hours of left anterior descending artery occlusion and slow reperfusion through a residual stenosis. All dogs received 1.5 mCi of thallium-201 after 40 minutes of coronary occlusion. During occlusion and 2 hours of reperfusion, serial hemodynamic, blood flow and myocardial thallium-201 activity measurements were made. The relative thallium-201 gradient (normal zone minus ischemic zone activity when initial normal activity is expressed as 100%) during left anterior descending coronary occlusion was similar in all groups. Group I, 87 +/- 3%; Group II, 78 +/- 6%; Group III, 83 +/- 6% (p = NS). After 2 hours of either method of reperfusion, the final relative gradient had decreased to a similar level (Group II, 51 +/- 9%; Group III, 42 +/- 6%). These values were not significantly different from the final relative thallium-201 gradient seen in dogs undergoing a sustained 3 hour occlusion (Group I, 55 +/- 5%). After 2 hours of reperfusion, both methods of reflow were associated with similar degrees of ''no reflow.'' Transmural flows in the central ischemic zone were 89 +/- 10% of normal in Group II and 71 +/- 6% of normal in Group III after reperfusion, with both flows substantially higher than the relative thallium-201 activities in these dogs

  1. The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a

    Directory of Open Access Journals (Sweden)

    Mann Graham J

    2009-01-01

    Full Text Available Abstract Background CDKN2A/p16INK4a is frequently altered in human cancers and it is the most important melanoma susceptibility gene identified to date. p16INK4a inhibits pRb phosphorylation and induces cell cycle arrest, which is considered its main tumour suppressor function. Nevertheless, additional activities may contribute to the tumour suppressor role of p16INK4a and could help explain its specific association with melanoma predisposition. To identify such functions we conducted a yeast-two-hybrid screen for novel p16INK4a binding partners. Results We now report that p16INK4a interacts with the chromatin remodelling factor BRG1. We investigated the cooperative roles of p16INK4a and BRG1 using a panel of cell lines and a melanoma cell model with inducible p16INK4a expression and BRG1 silencing. We found evidence that BRG1 is not required for p16INK4a-induced cell cycle inhibition and propose that the p16INK4a-BRG1 complex regulates BRG1 chromatin remodelling activity. Importantly, we found frequent loss of BRG1 expression in primary and metastatic melanomas, implicating this novel p16INK4a binding partner as an important tumour suppressor in melanoma. Conclusion This data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.

  2. Retroviruses Hijack Chromatin Loops to Drive Oncogene Expression and Highlight the Chromatin Architecture around Proto-Oncogenic Loci

    Science.gov (United States)

    Pattison, Jillian M.; Wright, Jason B.; Cole, Michael D.

    2015-01-01

    The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene. PMID:25799187

  3. Epigenetic Alterations in Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Johannes eGräff

    2015-12-01

    Full Text Available Alzheimer’s disease (AD is the major cause of dementia in Western societies. It progresses asymptomatically during decades before being belatedly diagnosed when therapeutic strategies have become unviable. Although several genetic alterations have been associated with AD, the vast majority of AD cases do not show strong genetic underpinnings and are thus considered a consequence of non-genetic factors. Epigenetic mechanisms allow for the integration of long-lasting non-genetic inputs on specific genetic backgrounds, and recently, a growing number of epigenetic alterations in AD have been described. For instance, an accumulation of dysregulated epigenetic mechanisms in aging, the predominant risk factor of AD, might facilitate the onset of the disease. Likewise, mutations in several enzymes of the epigenetic machinery have been associated with neurodegenerative processes that are altered in AD such as impaired learning and memory formation. Genome-wide and locus-specific epigenetic alterations have also been reported, and several epigenetically dysregulated genes validated by independent groups. From these studies, a picture emerges of AD as being associated with DNA hypermethylation and histone deacetylation, suggesting a general repressed chromatin state and epigenetically reduced plasticity in AD. Here we review these recent findings and discuss several technical and methodological considerations that are imperative for their correct interpretation. We also pay particular focus on potential implementations and theoretical frameworks that we expect will help to better direct future studies aimed to unravel the epigenetic participation in AD.

  4. Epigenetic Alterations in Alzheimer's Disease.

    Science.gov (United States)

    Sanchez-Mut, Jose V; Gräff, Johannes

    2015-01-01

    Alzheimer's disease (AD) is the major cause of dementia in Western societies. It progresses asymptomatically during decades before being belatedly diagnosed when therapeutic strategies have become unviable. Although several genetic alterations have been associated with AD, the vast majority of AD cases do not show strong genetic underpinnings and are thus considered a consequence of non-genetic factors. Epigenetic mechanisms allow for the integration of long-lasting non-genetic inputs on specific genetic backgrounds, and recently, a growing number of epigenetic alterations in AD have been described. For instance, an accumulation of dysregulated epigenetic mechanisms in aging, the predominant risk factor of AD, might facilitate the onset of the disease. Likewise, mutations in several enzymes of the epigenetic machinery have been associated with neurodegenerative processes that are altered in AD such as impaired learning and memory formation. Genome-wide and locus-specific epigenetic alterations have also been reported, and several epigenetically dysregulated genes validated by independent groups. From these studies, a picture emerges of AD as being associated with DNA hypermethylation and histone deacetylation, suggesting a general repressed chromatin state and epigenetically reduced plasticity in AD. Here we review these recent findings and discuss several technical and methodological considerations that are imperative for their correct interpretation. We also pay particular focus on potential implementations and theoretical frameworks that we expect will help to better direct future studies aimed to unravel the epigenetic participation in AD.

  5. Dedifferentiation of tobacco cells is associated with ribosomal RNA gene hypomethylation, increased transcription, and chromatin alterations

    Czech Academy of Sciences Publication Activity Database

    Koukalová, Blažena; Fojtová, Miloslava; Lim, Yoong Kar; Fulneček, Jaroslav; Leitch, Rowland Andrew; Kovařík, Aleš

    2005-01-01

    Roč. 139, - (2005), s. 275-286 ISSN 0032-0889 Institutional research plan: CEZ:AV0Z50040507 Keywords : pluripotent tobacco cells * epigenetic changes Subject RIV: BO - Biophysics Impact factor: 6.114, year: 2005

  6. DNA-repair, chromosome alterations and chromatin structure under environmental pollutions

    International Nuclear Information System (INIS)

    Altmann, H.

    1988-06-01

    54 abstracts, 20 of which are within the INIS scope, are presented. The papers are dealing with the influence of some chemicals, environmental pollutants as well as drugs, on the process of DNA repair after ionizing irradiation. Some advanced techniques of detecting genotoxic properties and some papers on the influence of DNA repair on cell differentiation were presented. Genetic changes in man, animals and plants as a consequence of the Chernobylsk accident were described

  7. Neutron scatter studies of chromatin structures related to functions

    International Nuclear Information System (INIS)

    Bradbury, E.M.

    1992-01-01

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

  8. Neutron scatter studies of chromatin structures related to functions

    International Nuclear Information System (INIS)

    Bradbury, E.M.

    1992-01-01

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

  9. Models of chromatin spatial organisation in the cell nucleus

    Science.gov (United States)

    Nicodemi, Mario

    2014-03-01

    In the cell nucleus chromosomes have a complex architecture serving vital functional purposes. Recent experiments have started unveiling the interaction map of DNA sites genome-wide, revealing different levels of organisation at different scales. The principles, though, which orchestrate such a complex 3D structure remain still mysterious. I will overview the scenario emerging from some classical polymer physics models of the general aspect of chromatin spatial organisation. The available experimental data, which can be rationalised in a single framework, support a picture where chromatin is a complex mixture of differently folded regions, self-organised across spatial scales according to basic physical mechanisms. I will also discuss applications to specific DNA loci, e.g. the HoxB locus, where models informed with biological details, and tested against targeted experiments, can help identifying the determinants of folding.

  10. Atomic force microscopy on chromosomes, chromatin and DNA: a review.

    Science.gov (United States)

    Kalle, Wouter; Strappe, Padraig

    2012-12-01

    The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  11. Encounter times of chromatin loci influenced by polymer decondensation

    Science.gov (United States)

    Amitai, A.; Holcman, D.

    2018-03-01

    The time for a DNA sequence to find its homologous counterpart depends on a long random search inside the cell nucleus. Using polymer models, we compute here the mean first encounter time (MFET) between two sites located on two different polymer chains and confined locally by potential wells. We find that reducing tethering forces acting on the polymers results in local decondensation, and numerical simulations of the polymer model show that these changes are associated with a reduction of the MFET by several orders of magnitude. We derive here new asymptotic formula for the MFET, confirmed by Brownian simulations. We conclude from the present modeling approach that the fast search for homology is mediated by a local chromatin decondensation due to the release of multiple chromatin tethering forces. The present scenario could explain how the homologous recombination pathway for double-stranded DNA repair is controlled by its random search step.

  12. Histone occurrence in chromatin from Peridinium balticum, a binucleate dinoflagellate.

    Science.gov (United States)

    Rizzo, P J; Cox, E R

    1977-12-23

    Peridinium balticum is one of two dinoflagellates known to have dissimilar nuclei together in the same cell. One nucleus (dinokaryotic) has permanently condensed chromosomes, while the other (eukaryotic) does not have morphologically distinct chromosomes. Acid extracts of chromatin prepared from a mixture of dinokaryotic and eukaryotic nuclei and purified eukaryotic nuclei give four bands that co-migrate with four of the five histones from calf thymus when analyzed in urea-containing polyacrylamide gels.

  13. Light scattering measurements supporting helical structures for chromatin in solution.

    Science.gov (United States)

    Campbell, A M; Cotter, R I; Pardon, J F

    1978-05-01

    Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.

  14. [Neutron scatter studies of chromatin structure related to function

    International Nuclear Information System (INIS)

    Bradbury, E.M.

    1990-01-01

    This study is concerned with the application of neutron scatter techniques to the different structural states of nucleosomes and chromatin with the long term objective of understanding how the enormous lengths of DNA are folded into chromosomes. Micrococcal nuclease digestion kinetics have defined two subnucleosome particles; the chromatosome with 168 bp DNA, the histone octamer and one H1 and the nucleosome core particle with 146 bp DNA and the histone octamer. As will be discussed, the structure of the 146 bp DNA core particle is known in solution at low resolution from neutron scatter studies and in crystals. Based on this structure, the authors have a working model for the chromatosome and the mode of binding of H1. In order to define the structure of the nucleosome and also the different orders of chromatin structures they need to know the paths of DNA that link nucleosomes and the factors associated with chromosome functions that act on those DNA paths. The major region for this situation is the inherent variabilities in nucleosome DNA sequences, in the histone subtypes and their states of chemical modification and in the precise locations of nucleosomes. Such variabilities obscure the underlying principles that govern the packaging of DNA into the different structural states of nucleosomes and chromatin. The only way to elucidate these principles is to study the structures of nucleosomes and oligonucleosomes that are fully defined. They have largely achieved these objectives

  15. Chromatin organisation and cancer prognosis: a pan-cancer study.

    Science.gov (United States)

    Kleppe, Andreas; Albregtsen, Fritz; Vlatkovic, Ljiljana; Pradhan, Manohar; Nielsen, Birgitte; Hveem, Tarjei S; Askautrud, Hanne A; Kristensen, Gunnar B; Nesbakken, Arild; Trovik, Jone; Wæhre, Håkon; Tomlinson, Ian; Shepherd, Neil A; Novelli, Marco; Kerr, David J; Danielsen, Håvard E

    2018-03-01

    Chromatin organisation affects gene expression and regional mutation frequencies and contributes to carcinogenesis. Aberrant organisation of DNA has been correlated with cancer prognosis in analyses of the chromatin component of tumour cell nuclei using image texture analysis. As yet, the methodology has not been sufficiently validated to permit its clinical application. We aimed to define and validate a novel prognostic biomarker for the automatic detection of heterogeneous chromatin organisation. Machine learning algorithms analysed the chromatin organisation in 461 000 images of tumour cell nuclei stained for DNA from 390 patients (discovery cohort) treated for stage I or II colorectal cancer at the Aker University Hospital (Oslo, Norway). The resulting marker of chromatin heterogeneity, termed Nucleotyping, was subsequently independently validated in six patient cohorts: 442 patients with stage I or II colorectal cancer in the Gloucester Colorectal Cancer Study (UK); 391 patients with stage II colorectal cancer in the QUASAR 2 trial; 246 patients with stage I ovarian carcinoma; 354 patients with uterine sarcoma; 307 patients with prostate carcinoma; and 791 patients with endometrial carcinoma. The primary outcome was cancer-specific survival. In all patient cohorts, patients with chromatin heterogeneous tumours had worse cancer-specific survival than patients with chromatin homogeneous tumours (univariable analysis hazard ratio [HR] 1·7, 95% CI 1·2-2·5, in the discovery cohort; 1·8, 1·0-3·0, in the Gloucester validation cohort; 2·2, 1·1-4·5, in the QUASAR 2 validation cohort; 3·1, 1·9-5·0, in the ovarian carcinoma cohort; 2·5, 1·8-3·4, in the uterine sarcoma cohort; 2·3, 1·2-4·6, in the prostate carcinoma cohort; and 4·3, 2·8-6·8, in the endometrial carcinoma cohort). After adjusting for established prognostic patient characteristics in multivariable analyses, Nucleotyping was prognostic in all cohorts except for the prostate carcinoma

  16. Enrichment of HP1a on Drosophila chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain.

    Directory of Open Access Journals (Sweden)

    Nicole C Riddle

    2012-09-01

    Full Text Available Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP-chip analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs, HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low T(m downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

  17. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

    Directory of Open Access Journals (Sweden)

    Athale Chaitanya

    2004-11-01

    sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

  18. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

    Science.gov (United States)

    Bacher, Christian P; Reichenzeller, Michaela; Athale, Chaitanya; Herrmann, Harald; Eils, Roland

    2004-01-01

    with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus. PMID:15560848

  19. The Drosophila melanogaster CHD1 chromatin remodeling factor modulates global chromosome structure and counteracts HP1a and H3K9me2.

    Science.gov (United States)

    Bugga, Lakshmi; McDaniel, Ivy E; Engie, Liana; Armstrong, Jennifer A

    2013-01-01

    CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.

  20. The Drosophila melanogaster CHD1 chromatin remodeling factor modulates global chromosome structure and counteracts HP1a and H3K9me2.

    Directory of Open Access Journals (Sweden)

    Lakshmi Bugga

    Full Text Available CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.

  1. Aggregation of fragmented chromatin associated with the appearance of products of its nuclease treatment

    International Nuclear Information System (INIS)

    Lobanenkov, V.V.; Mironov, N.M.; Kupriyanova, E.I.; Shapot, V.S.

    1986-01-01

    Isolated cell nuclei were incubated with nucleases, and then the chromatin was extracted with a low-salt buffer. When degradation of the nuclear chromatin DNase I or micrococcal nuclease is intensified, solubilization of the deoxyribonucleoprotein (DNP) in low-salt buffer at first increases, reaching a maximum in the case of hydrolysis of 2-4% of the nuclear DNA, but after intensive treatment with nucleases, it decreases sharply. Soluble fragmented chromatin is aggregated during treatment with DNase I. The addition of exogenous products of nuclease treatment of isolated nuclei to a preparation of gelatinous chromatin induces its aggregation. Pretreatment of nuclear chromatin with RNase prevents the solubilization of DNP by solutions with low ionic strength. Certain experimental data obtained using rigorous nuclease treatment are discussed; for their interpretation it is necessary to consider the effect of aggregation of fragmented chromatin by products of its nuclease degradation

  2. A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene

    DEFF Research Database (Denmark)

    Singh, Hari R; Nardozza, Aurelio P; Möller, Ingvar R

    2017-01-01

    DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain......-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic...... cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation....

  3. Restoring chromatin after replication: How new and old histone marks come together

    DEFF Research Database (Denmark)

    Jasencakova, Zusana; Groth, Anja

    2010-01-01

    In dividing cells genome stability and function rely on faithful transmission of both DNA sequence and its organization into chromatin. In the course of DNA replication chromatin undergoes transient genome-wide disruption followed by restoration on new DNA. This involves tight coordination of DNA...... replication and chromatin assembly processes in time and space. Dynamic recycling and de novo deposition of histones are fundamental for chromatin restoration. Histone post-translational modifications (PTMs) are thought to have a causal role in establishing distinct chromatin structures. Here we discuss PTMs...... present on new and parental histones and how they influence genome stability and restoration of epigenetically defined domains. Newly deposited histones must change their signature in the process of chromatin restoration, this may occur in a step-wise fashion involving replication-coupled processes...

  4. Autodigestion of chromatin in some radiosensitive and radioresistant mouse cells. Role of proteolysis and endonucleolysis

    International Nuclear Information System (INIS)

    Suciu, D.; Bojan, O.

    1981-01-01

    Evidence is presented indicating that mouse thymus, spleen, kidney, lung and heart contain a protease activity with relatively high specificity for histones. It is suggested that degradation of chromatin occurring in irradiated lymphoid tissues is produced by the action of alkaline endonuclease in association with this histone protease. The autodigestion of chromatin was assessed by determining the release of soluble chromatin from cells suspended in sucrose media of low ionic strength. It was found that the protease inhibitors, phenylmethylsulphonyl fluoride and especially NaHSO 3 , were also capable of depressing the activity of alkaline endonuclease, the fragmentation of chromatin, and the release of soluble chromatin. The results suggest that the release of histones from irradiated lymphoid tissues cannot be considered as a determinant step in the fragmentation of DNA in chromatin. (author)

  5. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

  6. Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization

    Directory of Open Access Journals (Sweden)

    Vuthy Ea

    2015-07-01

    Full Text Available Recent investigations on 3D chromatin folding revealed that the eukaryote genomes are both highly compartmentalized and extremely dynamic. This review presents the most recent advances in topological domains’ organization of the eukaryote genomes and discusses the relationship to chromatin loop formation. CTCF protein appears as a central factor of these two organization levels having either a strong insulating role at TAD borders, or a weaker architectural role in chromatin loop formation. TAD borders directly impact on chromatin dynamics by restricting contacts within specific genomic portions thus confining chromatin loop formation within TADs. We discuss how sub-TAD chromatin dynamics, constrained into a recently described statistical helix conformation, can produce functional interactions by contact stabilization.

  7. Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

    OpenAIRE

    Guo, Li; Feng, Yingang; Zhang, Zhenfeng; Yao, Hongwei; Luo, Yuanming; Wang, Jinfeng; Huang, Li

    2007-01-01

    Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a high...

  8. Scaffold Attachment Factor B1: A Novel Chromatin Regulator of Prostate Cancer Metabolism

    Science.gov (United States)

    2016-10-01

    AWARD NUMBER: W81XWH-14-1-0152 TITLE: Scaffold Attachment Factor B1: A Novel Chromatin Regulator of Prostate Cancer Metabolism PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-14-1-0152 Scaffold Attachment Factor B1: A Novel Chromatin Regulator of Prostate Cancer Metabolism... chromatin immunoprecipitation-next generation DNA sequencing (ChIP-seq) and integrative network modeling to identify the SAFB1 cistrome and the extent of

  9. Transcriptional decomposition reveals active chromatin architectures and cell specific regulatory interactions

    DEFF Research Database (Denmark)

    Rennie, Sarah; Dalby, Maria; van Duin, Lucas

    2018-01-01

    Transcriptional regulation is tightly coupled with chromosomal positioning and three-dimensional chromatin architecture. However, it is unclear what proportion of transcriptional activity is reflecting such organisation, how much can be informed by RNA expression alone and how this impacts disease...... proportion of total levels and is highly informative of topological associating domain activities and organisation, revealing boundaries and chromatin compartments. Furthermore, expression data alone accurately predict individual enhancer-promoter interactions, drawing features from expression strength...... between transcription and chromatin architecture....

  10. Chromatin association of UHRF1 during the cell cycle

    KAUST Repository

    Al-Gashgari, Bothayna

    2017-05-01

    Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a nuclear protein that associates with chromatin. Regardless of the various functions of UHRF1 in the cell, one of its more important functions is its role in the maintenance of DNA methylation patterns by the recruitment of DNMT1. Studies on UHRF1 based on this function have revealed the importance of UHRF1 during the cell cycle. Moreover, based on different studies various factors were described to be involved in the regulation of UHRF1 with different functionalities that can control its binding affinity to different targets on chromatin. These factors are regulated differently in a cell cycle specific manner. In light of this, we propose that UHRF1 has different binding behaviors during the cell cycle in regard to its association with chromatin. In this project, we first analyzed the binding behavior of endogenous UHRF1 from different unsynchronized cell systems in pull-down assays with peptides and oligonucleotides. Moreover, to analyze UHRF1 binding behavior during the cell cycle, we used two different approaches. First we sorted Jurkat and HT1080 cells based on their cell cycle stage using FACS analysis. Additionally, we synchronized HeLa cells to different stages of the cell cycle by chemical treatments, and used extracts from cellsorting and cell synchronization experiments for pull-down assays. We observed that UHRF1 in different cell systems has different preferences in regard to its binding to H3 unmodified and H3K9me3. Moreover, we detected that UHRF1, in general, displays different patterns between different stages of cell cycle; however, we cannot draw a final model for UHRF1 binding pattern during cell cycle.

  11. Analysis of Myc-induced histone modifications on target chromatin.

    Directory of Open Access Journals (Sweden)

    Francesca Martinato

    Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.

  12. Low Dose Radiation-Induced Genome and Epigenome Instability Symposium and Epigenetic Mechanisms, DNA Repair, and Chromatin Symposium at the EMS 2008 Annual Meeting - October 2008

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, William F; Kovalchuk, Olga; Dolinoy, Dana C; Dubrova, Yuri E; Coleman, Matthew A; Schär, Primo; Pogribny, Igor; Hendzel, Michael

    2010-02-19

    The Low Dose Radiation Symposium thoughtfully addressed ionizing radiation non-mutational but transmissable alterations in surviving cells. Deregulation of epigenetic processes has been strongly implicated in carcinogenesis, and there is increasing realization that a significant fraction of non-targeted and adaptive mechanisms in response to ionizing radiation are likely to be epigenetic in nature. Much remains to be learned about how chromatin and epigenetic regulators affect responses to low doses of radiation, and how low dose radiation impacts other epigenetic processes. The Epigenetic Mechanisms Symposium focused on on epigenetic mechanisms and their interplay with DNA repair and chromatin changes. Addressing the fact that the most well understood mediators of epigenetic regulation are histone modifications and DNA methylation. Low levels of radiation can lead to changes in the methylation status of certain gene promoters and the expression of DNA methyltransferases, However, epigenetic regulation can also involve changes in higher order chromosome structure.

  13. Chromatin structure and epigenetics of tumour cells: A review

    Czech Academy of Sciences Publication Activity Database

    Bártová, Eva; Krejčí, Jana; Hájek, R.; Harničarová, Andrea; Kozubek, Stanislav

    2009-01-01

    Roč. 9, č. 1 (2009), s. 51-61 ISSN 1871-529X R&D Projects: GA AV ČR(CZ) 1QS500040508; GA ČR(CZ) GA204/06/0978 Grant - others:GA MŠk(CZ) LC06027; GA MŠk(CZ) LC535 Program:LC; LC Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : tumour cells * chromatin * radiation Subject RIV: BO - Biophysics

  14. Keeping it quiet: chromatin control of gammaherpesvirus latency.

    Science.gov (United States)

    Lieberman, Paul M

    2013-12-01

    The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) establish long-term latent infections associated with diverse human cancers. Viral oncogenesis depends on the ability of the latent viral genome to persist in host nuclei as episomes that express a restricted yet dynamic pattern of viral genes. Multiple epigenetic events control viral episome generation and maintenance. This Review highlights some of the recent findings on the role of chromatin assembly, histone and DNA modifications, and higher-order chromosome structures that enable gammaherpesviruses to establish stable latent infections that mediate viral pathogenesis.

  15. Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

    Science.gov (United States)

    Dillinger, Stefan; Németh, Attila

    2016-01-01

    The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

  16. Genomic and chromatin signals underlying transcription start-site selection

    DEFF Research Database (Denmark)

    Valen, Eivind; Sandelin, Albin Gustav

    2011-01-01

    A central question in cellular biology is how the cell regulates transcription and discerns when and where to initiate it. Locating transcription start sites (TSSs), the signals that specify them, and ultimately elucidating the mechanisms of regulated initiation has therefore been a recurrent theme....... In recent years substantial progress has been made towards this goal, spurred by the possibility of applying genome-wide, sequencing-based analysis. We now have a large collection of high-resolution datasets identifying locations of TSSs, protein-DNA interactions, and chromatin features over whole genomes...

  17. Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli.

    Science.gov (United States)

    Stępiński, Dariusz

    2013-06-01

    Nucleolar chromatin, including nucleolus-associated chromatin as well as active and inactive condensed ribosomal DNA (rDNA) chromatin, derives mostly from secondary constrictions known as nucleolus organizer regions containing rDNA genes on nucleolus-forming chromosomes. This chromatin may occupy different nucleolar positions being in various condensation states which may imply different rDNA transcriptional competence. Sections of nucleoli originating from root meristematic cells of soybean seedlings grown at 25 °C (the control), then subjected to chilling stress (10 °C), and next transferred again to 25 °C (the recovery) were used to measure profile areas occupied by nucleolar condensed chromatin disclosed with sodium hydroxide methylation-acetylation plus uranyl acetate technique. The biggest total area of condensed chromatin was found in the nucleoli of chilled plants, while the smallest was found in those of recovered plants in relation to the amounts of chromatin in the control nucleoli. The condensed nucleolar chromatin, in the form of different-sized and different-shaped clumps, was mainly located in fibrillar centers. One can suppose that changes of condensed rDNA chromatin amounts might be a mechanism controlling the number of transcriptionally active rDNA genes as the nucleoli of plants grown under these experimental conditions show different transcriptional activity and morphology.

  18. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    DEFF Research Database (Denmark)

    Düring, Louis; Thorsen, Michael; Petersen, Darima

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

  19. Extracellular Matrix-Induced Chromatin Modifications in Normal and Malignant Human Breast Cells

    National Research Council Canada - National Science Library

    Beyec, Johanne

    2002-01-01

    .... The changes in gene expression that occur during differentiation or tumorigenesis are accompanied by characteristics patterns of chromatin reorganization, modulated, in part, through highly regulated...

  20. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    Directory of Open Access Journals (Sweden)

    Shieh Grace S

    2011-12-01

    Full Text Available Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1 marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3, which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  1. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    LENUS (Irish Health Repository)

    Shieh, Grace S.

    2011-12-22

    Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3\\' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  2. Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription

    Directory of Open Access Journals (Sweden)

    Habiba Harouz

    2014-12-01

    Full Text Available Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector “uses” HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014. OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up.

  3. Comparative aspects of basic chromatin proteins in dinoflagellates.

    Science.gov (United States)

    Rizzo, P J

    1981-01-01

    Previous work on histone-like proteins in dinoflagellates is summarized, together with some new data to give an overview of basic proteins in these algae. The first two dinoflagellates studied were both found to contain one major acid-soluble protein that migrated to the same position in acidic-urea gels. When several other genera were studied however, it became apparent that the histone-like proteins from different dinoflagellates were similar but not identical. In view of the great diversity of living dinoflagellates it is speculated that further differences in dinoflagellate basic chromatin proteins will be revealed. Electrophoretic data from the eukaryotic (endosymbiont) nucleus of Peridinium balticum showed the presence of five major components. It is speculated that two of these proteins represent an H1-like doublet and two others correspond to the highly conserved histones H3 and H4. The fifth component is a new histone that may substitute for H2A and H2B in the nucleosome. Because histones and nucleosomes are present in all higher organisms but completely lacking in procaryotes, studies on basic proteins in dinoflagellates will provides insights into the evolution of histones and eucaryotic chromatin organization.

  4. Synaptic, transcriptional and chromatin genes disrupted in autism.

    Science.gov (United States)

    De Rubeis, Silvia; He, Xin; Goldberg, Arthur P; Poultney, Christopher S; Samocha, Kaitlin; Cicek, A Erucment; Kou, Yan; Liu, Li; Fromer, Menachem; Walker, Susan; Singh, Tarinder; Klei, Lambertus; Kosmicki, Jack; Shih-Chen, Fu; Aleksic, Branko; Biscaldi, Monica; Bolton, Patrick F; Brownfeld, Jessica M; Cai, Jinlu; Campbell, Nicholas G; Carracedo, Angel; Chahrour, Maria H; Chiocchetti, Andreas G; Coon, Hilary; Crawford, Emily L; Curran, Sarah R; Dawson, Geraldine; Duketis, Eftichia; Fernandez, Bridget A; Gallagher, Louise; Geller, Evan; Guter, Stephen J; Hill, R Sean; Ionita-Laza, Juliana; Jimenz Gonzalez, Patricia; Kilpinen, Helena; Klauck, Sabine M; Kolevzon, Alexander; Lee, Irene; Lei, Irene; Lei, Jing; Lehtimäki, Terho; Lin, Chiao-Feng; Ma'ayan, Avi; Marshall, Christian R; McInnes, Alison L; Neale, Benjamin; Owen, Michael J; Ozaki, Noriio; Parellada, Mara; Parr, Jeremy R; Purcell, Shaun; Puura, Kaija; Rajagopalan, Deepthi; Rehnström, Karola; Reichenberg, Abraham; Sabo, Aniko; Sachse, Michael; Sanders, Stephan J; Schafer, Chad; Schulte-Rüther, Martin; Skuse, David; Stevens, Christine; Szatmari, Peter; Tammimies, Kristiina; Valladares, Otto; Voran, Annette; Li-San, Wang; Weiss, Lauren A; Willsey, A Jeremy; Yu, Timothy W; Yuen, Ryan K C; Cook, Edwin H; Freitag, Christine M; Gill, Michael; Hultman, Christina M; Lehner, Thomas; Palotie, Aaarno; Schellenberg, Gerard D; Sklar, Pamela; State, Matthew W; Sutcliffe, James S; Walsh, Christiopher A; Scherer, Stephen W; Zwick, Michael E; Barett, Jeffrey C; Cutler, David J; Roeder, Kathryn; Devlin, Bernie; Daly, Mark J; Buxbaum, Joseph D

    2014-11-13

    The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

  5. Chromatin Structure and Radiation-Induced Intrachromosome Exchange

    Science.gov (United States)

    Mangala; Zhang, Ye; Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    2011-01-01

    We have recently investigated the location of breaks involved in intrachromosomal type exchange events, using the multicolor banding in situ hybridization (mBAND) technique for human chromosome 3. In human epithelial cells exposed to both low- and high-LET radiations in vitro, intrachromosome exchanges were found to occur preferentially between a break in the 3p21 and one in the 3q11. Exchanges were also observed between a break in 3p21 and one in 3q26, but few exchanges were observed between breaks in 3q11 and 3q26, even though the two regions were on the same arm of the chromosome. To explore the relationships between intrachromosome exchanges and chromatin structure, we used probes that hybridize the three regions of 3p21, 3q11 and 3q26, and measured the distance between two of the three regions in interphase cells. We further analyzed fragile sites on the chromosome that have been identified in various types of cancers. Our results demonstrated that the distribution of breaks involved in radiation-induced intrachromosome aberrations depends upon both the location of fragile sites and the folding of chromatins

  6. The role of proteins and metal ions in the protection of chromatin DNA at fast neutrons action

    International Nuclear Information System (INIS)

    Radu, L.; Preoteasa, V.; Radulescu, I.; Constantinescu, B.

    1997-01-01

    The role of chromatin proteins and of some ions on the fast neutrons actions on chromatin DNA from rat Walker tumors was analysed. The DNA in chromatin is effectively protected against fast neutrons actions by DNA bound proteins and specially by histones, because of the limited accessibility of the condensed chromatin DNA to hydroxyl radicals and of the scavenging of radicals by the chromatin proteins. The ions utilised protect chromatin DNA against the damage produced ed by fast neutrons, through the induction of structural DNA changes with a less accessibility to OH radicals. (authors)

  7. Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

    Directory of Open Access Journals (Sweden)

    Mora Xavier

    2007-05-01

    Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

  8. Distribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatin

    International Nuclear Information System (INIS)

    Smerdon, M.J.; Tlsty, T.D.; Lieberman, M.W.

    1978-01-01

    The distribution of ultraviolet radiation (uv) induced DNA repair synthesis within chromatin was examined in cultured human diploid fibroblasts (IMR-90). Measurement of the time course of repair synthesis yielded two distinct phases: An initial rapid phase (fast repair) which occurs during the first 2 to 3 h after damage and a slower phase (slow repair) associated with a tenfold decrease in the rate of nucleotide incorporation, which persists for at least 35 h after damage. Staphylococcal nuclease digests of nuclei from cells damaged with uv and labeled during the fast-repair phase revealed a marked preference of fast-repair synthesis for the nuclease-sensitive regions. A new method was developed to analyze the digestion data and showed that approximately 50% of the nucleotides incorporated during the fast-repair phase are located in staphylococcal nuclease-sensitive regions, which comprise about 30% of the genome. Calculations from these data indicate that in the staphylococcal nuclease-sensitive regions the number of newly inserted nucleotides per unit DNA is about twice that of resistant regions. These results were supported by electrophoresis studies which demonstrated a decreased representation of fast-repair synthesis in core particle DNA. In contrast, the distribution within chromatin of nucleotides incorporated during the slow-repair phase was found to be much more homogeneous with about 30% of the repair sites located in 25% of the genome. Digestion studieswith DNase I indicated a slight preference of repair synthesis for regions sensitive to this enzyme; however, no marked difference between the distributions of fast- and slow-repair synthesis was observed. This study provides evidence that the structural constraints placed upon DNA in chromatin also place constraints upon uv-induced DNA repair synthesis in human cells

  9. Lymphocytes From Patients With Type 1 Diabetes Display a Distinct Profile of Chromatin Histone H3 Lysine 9 Dimethylation

    Science.gov (United States)

    Miao, Feng; Smith, David D.; Zhang, Lingxiao; Min, Andrew; Feng, Wei; Natarajan, Rama

    2008-01-01

    OBJECTIVE—The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications. RESEARCH DESIGN AND METHODS—We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools. RESULTS—A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-β, nuclear factor-κB, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes. CONCLUSIONS—The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes–related biological pathways and also a new

  10. Neuron-specific chromatin remodeling: a missing link in epigenetic mechanisms underlying synaptic plasticity, memory, and intellectual disability disorders.

    Science.gov (United States)

    Vogel-Ciernia, Annie; Wood, Marcelo A

    2014-05-01

    Long-term memory formation requires the coordinated regulation of gene expression. Until recently nucleosome remodeling, one of the major epigenetic mechanisms for controlling gene expression, had been largely unexplored in the field of neuroscience. Nucleosome remodeling is carried out by chromatin remodeling complexes (CRCs) that interact with DNA and histones to physically alter chromatin structure and ultimately regulate gene expression. Human exome sequencing and gene wide association studies have linked mutations in CRC subunits to intellectual disability disorders, autism spectrum disorder and schizophrenia. However, how mutations in CRC subunits were related to human cognitive disorders was unknown. There appears to be both developmental and adult specific roles for the neuron specific CRC nBAF (neuronal Brg1/hBrm Associated Factor). nBAF regulates gene expression required for dendritic arborization during development, and in the adult, contributes to long-term potentiation, a form of synaptic plasticity, and long-term memory. We propose that the nBAF complex is a novel epigenetic mechanism for regulating transcription required for long-lasting forms of synaptic plasticity and memory processes and that impaired nBAF function may result in human cognitive disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

    Science.gov (United States)

    Sanborn, Adrian; Rao, Suhas; Huang, Su-Chen; Durand, Neva; Huntley, Miriam; Jewett, Andrew; Bochkov, Ivan; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher; McKenna, Doug; Stamenova, Elena; Gnirke, Andreas; Melnikov, Alexandre; Lander, Eric; Aiden, Erez

    Our recent kilobase-resolution genome-wide maps of DNA self-contacts demonstrated that mammalian genomes are organized into domains and loops demarcated by the DNA-binding protein CTCF. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that domains are inconsistent with equilibrium and fractal models. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during condensation leads to formation of an anisotropic ``tension globule.'' In the other, CTCF and cohesin act together to extrude unknotted loops. Both models are consistent with the observed domains and loops. However, the extrusion model explains a far wider array of observations, such as why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The extrusion model predicts in silico the experimental maps using only CTCF-binding sites. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

  12. Gibberellin-induced change in the structure of chromatin in wheat sprouts: decrease in the accessibility of DNA in preparations of soluble chromatin to the action of EcoRII methylase

    International Nuclear Information System (INIS)

    Noskov, V.A.; Kintsurashvili, L.N.; Smirnova, T.A.; Manamsh'yan, T.A.; Kir'yanov, G.I.; Vanyushin, B.F.

    1986-01-01

    A method has been perfected for producing soluble chromatin from whole wheat sprouts at low ionic strength. The chromatin preparations isolated possess a native structure: they have a nucleosome organization. Under identical conditions the soluble wheat chromatin undergoes more profound degradation by DNase I and staphylococcal nuclease than the chromatin from the rat liver. The DNA contained in the isolated chromatin is capable of accepting CHnumber groups from S-[methyl- 3 H]-adenosylmethionine during incubation with DNA methylase EcoRII; not all the CC A/T GG sequences in DNA are methylated in vivo. Chromatin from gibberellin A 3 -treated wheat sprout DNA accepts 40% fewer CH 3 groups than that from the control sprouts, which is probably due to the greater compactness of the chromatin. In the case of longer incubation, the level of methylation of the chromatin falls, which may be associated with the presence of DNA-demethylating activity

  13. Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

    Science.gov (United States)

    Hulsman, Marc; Lelieveldt, Boudewijn P. F.; de Ridder, Jeroen; Reinders, Marcel

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequent long-range chromatin interactions remains challenging. We address this by characterizing the topology of the cortical chromatin interaction network using scale-aware topological measures. We demonstrate that based on these characterizations it is possible to accurately predict spatial co-expression between genes in the mouse cortex. Consistent with previous findings, we find that the chromatin interaction profile of a gene-pair is a good predictor of their spatial co-expression. However, the accuracy of the prediction can be substantially improved when chromatin interactions are described using scale-aware topological measures of the multi-resolution chromatin interaction network. We conclude that, for co-expression prediction, it is necessary to take into account different levels of chromatin interactions ranging from direct interaction between genes (i.e. small-scale) to chromatin compartment interactions (i.e. large-scale). PMID:25965262

  14. Regular character of chromatin degradation in lymphoid tissues after treatment with biological alkylating agents in vivo

    International Nuclear Information System (INIS)

    Matyasova, J.; Skalka, M.; Cejkova, M.

    1979-01-01

    The chromatin changes are reevaluated occurring in lymphoid tissues of mice treated with alkylating agents of the nitrogen-mustard type in relation to recent evidence on the nucleosomal organization of chromatin and to our new data on the regular character of chromatin degradation in lymphoid tissues of irradiated mice. DNA was isolated from nuclei at various intervals (1 to 18 h) after treatment of mice and subjected to gel electrophoresis in polyacrylamide gels. Thymus chromatin from treated mice has been shown to degrade in a regular fashion and to yield discrete DNA fragments, resembling those that originate in lymphoid tissues of irradiated mice or in thymus nuclei digested with micrococcal nuclease in vitro. With increasing interval after treatment higher amounts of smaller DNA fragments appear. Chromatin in spleen cells responds to treatment in a similar way, whilst no degradation in vivo takes place in liver chromatin. Chromatin of LS/BL lymphosarcoma cells in mice treated with alkylating agents or with irradiation suffers from a similar regular degradation. The results stress the significance of the action of liberated or activated endogenous nuclease(s) in the development of chromatin damage in lymphoid cells after treatment with alkylating agents. (author)

  15. Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

    NARCIS (Netherlands)

    Boyer, L.A.; Logie, C.; Bonte, E; Becker, P.B.; Wade, P.A.; Wolff, A.P.; Wu, C.; Imbalzano, A.N.; Peterson, C.L.

    2000-01-01

    ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates.

  16. Reading the maps: Organization and function of chromatin types in Drosophila

    NARCIS (Netherlands)

    Braunschweig, U.

    2010-01-01

    The work presented in this thesis shows that the Drosophila genome is organized in chromatin domains with many implications for gene regulation, nuclear organization, and evolution. Furthermore it provides examples of how maps of chromatin protein binding, combined with computational approaches, can

  17. Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco

    NARCIS (Netherlands)

    Mlynárová, L.; Loonen, A.; Mietkiewska, E.; Jansen, R.C.; Nao, J.P.

    2002-01-01

    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli ß-glucuronidase gene and the firefly luciferase gene, driven by different

  18. Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco

    NARCIS (Netherlands)

    Mlynárová, Ludmila; Loonen, Annelies; Mietkiewska, Elzbieta; Jansen, Ritsert C.; Nap, Jan-Peter

    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli β-glucuronidase gene and the firefly luciferase gene, driven by different

  19. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

    NARCIS (Netherlands)

    T.B. van Dijk (Thamar); N. Gillemans (Nynke); C. Stein (Claudia); P. Fanis (Pavlos); J.A.A. Demmers (Jeroen); M.P.C. van de Corput (Mariëtte); J. Essers (Jeroen); F.G. Grosveld (Frank); U.M. Bauer (Uta-Maria); J.N.J. Philipsen (Sjaak)

    2010-01-01

    textabstractWe describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified

  20. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity

    Directory of Open Access Journals (Sweden)

    Tahereh Rahiminia

    2017-08-01

    Conclusion: Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome integrity were decreased in Vit. However, these findings were more acceptable in SSV or Vapour.

  1. High-throughput assessment of context-dependent effects of chromatin proteins

    NARCIS (Netherlands)

    Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

    2016-01-01

    textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

  2. Small chromosomal regions position themselves autonomously according to their chromatin class.

    Science.gov (United States)

    van de Werken, Harmen J G; Haan, Josien C; Feodorova, Yana; Bijos, Dominika; Weuts, An; Theunis, Koen; Holwerda, Sjoerd J B; Meuleman, Wouter; Pagie, Ludo; Thanisch, Katharina; Kumar, Parveen; Leonhardt, Heinrich; Marynen, Peter; van Steensel, Bas; Voet, Thierry; de Laat, Wouter; Solovei, Irina; Joffe, Boris

    2017-06-01

    The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes. © 2017 van de Werken et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Prediction of highly expressed genes in microbes based on chromatin accessibility

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2007-01-01

    BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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. (perspective)

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

    Science.gov (United States)

    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.

  6. Toxic effects of lead and nickel nitrate on rat liver chromatin components.

    Science.gov (United States)

    Rabbani-Chadegani Iii, Azra; Fani, Nesa; Abdossamadi, Sayeh; Shahmir, Nosrat

    2011-01-01

    The biological activity of heavy metals is related to their physicochemical interaction with biological receptors. In the present study, the effect of low concentrations of nickel nitrate and lead nitrate (lead nitrate to chromatin compared to nickel nitrate. Also, the binding affinity of lead nitrate to histone proteins free in solution was higher than nickel. On the basis of the results, it is concluded that lead reacts with chromatin components even at very low concentrations and induce chromatin aggregation through histone-DNA cross-links. Whereas, nickel nitrate is less effective on chromatin at low concentrations, suggesting higher toxicity of lead nitrate on chromatin compared to nickel. Copyright © 2010 Wiley Periodicals, Inc.

  7. Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Hangnoh Lee

    2016-09-01

    Full Text Available Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

  8. Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

    Science.gov (United States)

    Lee, Hangnoh; Cho, Dong-Yeon; Whitworth, Cale; Eisman, Robert; Phelps, Melissa; Roote, John; Kaufman, Thomas; Cook, Kevin; Russell, Steven; Przytycka, Teresa; Oliver, Brian

    2016-09-01

    Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

  9. An overproduction of astellolides induced by genetic disruption of chromatin-remodeling factors in Aspergillus oryzae.

    Science.gov (United States)

    Shinohara, Yasutomo; Kawatani, Makoto; Futamura, Yushi; Osada, Hiroyuki; Koyama, Yasuji

    2016-01-01

    The filamentous fungus Aspergillus oryzae is an important industrial mold. Recent genomic analysis indicated that A. oryzae has a large number of biosynthetic genes for secondary metabolites (SMs), but many of the SMs they produce have not been identified. For better understanding of SMs production by A. oryzae, we screened a gene-disruption library of transcription factors including chromatin-remodeling factors and found two gene disruptions that show similarly altered SM production profiles. One is a homolog of Aspergillus nidulans cclA, a component of the histone 3 lysine 4 (H3K4) methyltransferase complex of proteins associated with Set1 complex, and the other, sppA, is an ortholog of Saccharomyces cerevisiae SPP1, another component of a complex of proteins associated with Set1 complex. The cclA and sppA disruptions in A. oryzae are deficient in trimethylation of H3K4. Furthermore, one of the SMs that increased in the cclA disruptant was identified as astellolide F (14-deacetyl astellolide B). These data indicate that both cclA and sppA affect production of SMs including astellolides by affecting the methylation status of H3K4 in A. oryzae.

  10. Smectite alteration

    International Nuclear Information System (INIS)

    Anderson, D.M.

    1984-11-01

    This report contains the proceedings of a second workshop in Washington DC December 8-9, 1983 on the alteration of smectites intended for use as buffer materials in the long-term containment of nuclear wastes. It includes extended summaries of all presentations and a transcript of the detailed scientific discussion. The discussions centered on three main questions: What is the prerequisite for and what is the precise mechanism by which smectite clays may be altered to illite. What are likly sources of potassium with respect to the KBS project. Is it likely that the conversion of smectite to illite will be of importance in the 10 5 to the 10 6 year time frame. The workshop was convened to review considerations and conclusions in connection to these questions and also to broaden the discussion to consider the use of smectite clays as buffer materials for similar applications in different geographical and geological settings. SKBF/KBS technical report 83-03 contains the proceedings from the first workshop on these matters that was held at the State University of New York, Buffalo May 26-27, 1982. (Author)

  11. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    Directory of Open Access Journals (Sweden)

    Christopher Ian Cazzonelli

    2014-10-01

    Full Text Available Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3, which encodes a calmodulin-like protein (CML12. The gene neighbouring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

  12. Selection on a Subunit of the NURF Chromatin Remodeler Modifies Life History Traits in a Domesticated Strain of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Edward E Large

    2016-07-01

    Full Text Available Evolutionary life history theory seeks to explain how reproductive and survival traits are shaped by selection through allocations of an individual's resources to competing life functions. Although life-history traits evolve rapidly, little is known about the genetic and cellular mechanisms that control and couple these tradeoffs. Here, we find that two laboratory-adapted strains of C. elegans descended from a single common ancestor that lived in the 1950s have differences in a number of life-history traits, including reproductive timing, lifespan, dauer formation, growth rate, and offspring number. We identified a quantitative trait locus (QTL of large effect that controls 24%-75% of the total trait variance in reproductive timing at various timepoints. Using CRISPR/Cas9-induced genome editing, we show this QTL is due in part to a 60 bp deletion in the 3' end of the nurf-1 gene, which is orthologous to the human gene encoding the BPTF component of the NURF chromatin remodeling complex. Besides reproduction, nurf-1 also regulates growth rate, lifespan, and dauer formation. The fitness consequences of this deletion are environment specific-it increases fitness in the growth conditions where it was fixed but decreases fitness in alternative laboratory growth conditions. We propose that chromatin remodeling, acting through nurf-1, is a pleiotropic regulator of life history trade-offs underlying the evolution of multiple traits across different species.

  13. Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

    2009-06-03

    Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

  14. A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation.

    Science.gov (United States)

    Cazzonelli, Christopher I; Nisar, Nazia; Roberts, Andrea C; Murray, Kevin D; Borevitz, Justin O; Pogson, Barry J

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

  15. Time-resolved spectroscopy and fluorescence resonance energy transfer in the study of excimer laser damage of chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [Department of Molecular Genetics and Radiobiology, Babes National Institute, Bucharest (Romania)], E-mail: lilianajradu@yahoo.fr; Mihailescu, I. [Department of Lasers, Laser, Plasma and Radiation Physics Institute, Bucharest (Romania); Radu, S. [Department of Computer Science, Polytechnics University, Bucharest (Romania); Gazdaru, D. [Department of Biophysics, Bucharest University (Romania)

    2007-09-21

    The analysis of chromatin damage produced by a 248 nm excimer laser radiation, for doses of 0.3-3 MJ/m{sup 2} was carried out by time-resolved spectroscopy and fluorescence resonance energy transfer (FRET). The chromatin was extracted from a normal and a tumoral tissue of Wistar rats. The decrease with laser dose of the relative contribution of the excited state lifetimes of ethidium bromide (EtBr) bounded to chromatin constitutes an evidence of the reduction of chromatin deoxyribonucleic acid (DNA) double-strand structure. FRET was performed from dansyl chloride to acridine orange, both coupled to chromatin. The increase of the average distance between these ligands, under the action of laser radiation, reflects a loosening of the chromatin structure. The radiosensitivity of tumor tissue chromatin is higher than that of a normal tissue. The determination of the chromatin structure modification in an excimer laser field can be of interest in laser therapy.

  16. Time-resolved spectroscopy and fluorescence resonance energy transfer in the study of excimer laser damage of chromatin

    International Nuclear Information System (INIS)

    Radu, L.; Mihailescu, I.; Radu, S.; Gazdaru, D.

    2007-01-01

    The analysis of chromatin damage produced by a 248 nm excimer laser radiation, for doses of 0.3-3 MJ/m 2 was carried out by time-resolved spectroscopy and fluorescence resonance energy transfer (FRET). The chromatin was extracted from a normal and a tumoral tissue of Wistar rats. The decrease with laser dose of the relative contribution of the excited state lifetimes of ethidium bromide (EtBr) bounded to chromatin constitutes an evidence of the reduction of chromatin deoxyribonucleic acid (DNA) double-strand structure. FRET was performed from dansyl chloride to acridine orange, both coupled to chromatin. The increase of the average distance between these ligands, under the action of laser radiation, reflects a loosening of the chromatin structure. The radiosensitivity of tumor tissue chromatin is higher than that of a normal tissue. The determination of the chromatin structure modification in an excimer laser field can be of interest in laser therapy

  17. Snake-like chromatin in conjunctival cells of a population aged 30-60 years from Copenhagen City

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1998-01-01

    ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin......ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin...

  18. Elucidation of Chromatin Remodeling Machinery Involved in Regulation of Estrogen Receptor Alpha Expression in Human Breast Cancer Cells

    National Research Council Canada - National Science Library

    Sharma, Dipali

    2005-01-01

    .... Using chromatin immunoprecipitation (ChIP), we examined the chromatin status and repressor complex associated with silenced ER and changes in the key regulatory factors during reactivation by inhibitors of DNMT...

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

    Science.gov (United States)

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

    2016-09-26

    In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Transcriptional and chromatin regulation during fasting – The genomic era

    Science.gov (United States)

    Goldstein, Ido; Hager, Gordon L.

    2015-01-01

    An elaborate metabolic response to fasting is orchestrated by the liver and is heavily reliant upon transcriptional regulation. In response to hormones (glucagon, glucocorticoids) many transcription factors (TFs) are activated and regulate various genes involved in metabolic pathways aimed at restoring homeostasis: gluconeogenesis, fatty acid oxidation, ketogenesis and amino acid shuttling. We summarize the recent discoveries regarding fasting-related TFs with an emphasis on genome-wide binding patterns. Collectively, the summarized findings reveal a large degree of co-operation between TFs during fasting which occurs at motif-rich DNA sites bound by a combination of TFs. These new findings implicate transcriptional and chromatin regulation as major determinants of the response to fasting and unravels the complex, multi-TF nature of this response. PMID:26520657

  1. Human Transcriptome and Chromatin Modifications: An ENCODE Perspective

    Directory of Open Access Journals (Sweden)

    Li Shen

    2013-06-01

    Full Text Available A decade-long project, led by several international research groups, called the Encyclopedia of DNA Elements (ENCODE, recently released an unprecedented amount of data. The ambitious project covers transcriptome, cistrome, epigenome, and interactome data from more than 1,600 sets of experiments in human. To make use of this valuable resource, it is important to understand the information it represents and the techniques that were used to generate these data. In this review, we introduce the data that ENCODE generated, summarize the observations from the data analysis, and revisit a computational approach that ENCODE used to predict gene expression, with a focus on the human transcriptome and its association with chromatin modifications.

  2. Causes and consequences of chromatin variation between inbred mice.

    Directory of Open Access Journals (Sweden)

    Mona Hosseini

    2013-06-01

    Full Text Available Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS. Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10% of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.

  3. Chromatin Dynamics in Vivo: A Game of Musical Chairs

    Directory of Open Access Journals (Sweden)

    Daniël P. Melters

    2015-08-01

    Full Text Available Histones are a major component of chromatin, the nucleoprotein complex fundamental to regulating transcription, facilitating cell division, and maintaining genome integrity in almost all eukaryotes. In addition to canonical, replication-dependent histones, replication-independent histone variants exist in most eukaryotes. In recent years, steady progress has been made in understanding how histone variants assemble, their involvement in development, mitosis, transcription, and genome repair. In this review, we will focus on the localization of the major histone variants H3.3, CENP-A, H2A.Z, and macroH2A, as well as how these variants have evolved, their structural differences, and their functional significance in vivo.

  4. [Effect of irradiation on the degradation of rat thymocyte chromatin].

    Science.gov (United States)

    Tsudzevich, B O; Parkhomets', Iu P; Andriĭchuk, T R; Iurkina, V V

    1998-01-01

    Genome instability of adaptive nature is formed under the experimental influence on a cell. Under critical conditions, strategy of organism is to damage the cells that cannot be restored and controlled by including the program of apoptosis. The ordered internucleosomal DNA degradation is considered to be one of the proof attributes of immunocompetent cell apoptosis. We investigated the effects of various doses of irradiation on the thymocytes chromatine fragmentation in 1,2,3 hours after a single X-ray exposure or after chronic influence in conditions of Chernobyl research base. By the means of electrophoresis in agarose and judging by polydeoxyribonucleotides accumulation we observed the "ladder pattern" of degradation in 3 hr after single 1 Gr irradiation (the smallest dose displaying the effect). We suppose that the influence of both chronic low-intensity irradiation taking place in Chernobyl and single X-ray exposure result in intensifying of DNA fragmentation in the cells of immunocompetent organs.

  5. Chromatin Constrains the Initiation and Elongation of DNA Replication.

    Science.gov (United States)

    Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

    2017-01-05

    Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Chromatin in fractal globule state: evidence from comet assay

    Directory of Open Access Journals (Sweden)

    Afanasieva K. S.

    2015-04-01

    Full Text Available At higher order levels chromatin is organized into loops that appear as a result of contacts between distant loci. The aim of this work was to investigate the length distribution of the DNA loops in nucleoids obtained after lysis of either whole cells or isolated cell nuclei. Methods. We used single cell gel electrophoresis to analyze the kinetics of the DNA loop migration from the two nucleoid types. Results. The kinetics of the DNA exit was found to have specific features for the two types of nucleoids. At the same time, in both cases, the DNA amount in the electrophoretic track depends linearly on the length of the longest loops in the track. Conclusions. We have concluded that for the loops up to ~ 100 kb the length distribution appears to be consistent with the fractal globule organization.

  7. Chromatin dynamics in Pollen Mother Cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Wenjing eShe

    2015-04-01

    Full Text Available Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMCs committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition towards the male reproductive lineage. Here we show that Arabidopsis PMCs differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMCs. This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages.

  8. Comparative transmission genetics of introgressed chromatin in Gossypium (cotton) polyploids.

    Science.gov (United States)

    Waghmare, Vijay N; Rong, Junkang; Rogers, Carl J; Bowers, John E; Chee, Peng W; Gannaway, John R; Katageri, Ishwarappa; Paterson, Andrew H

    2016-04-01

    Introgression is widely acknowledged as a potential source of valuable genetic variation, and growing effort is being invested in analysis of interspecific crosses conferring transgressive variation. Experimental backcross populations provide an opportunity to study transmission genetics following interspecific hybridization, identifying opportunities and constraints to introgressive crop improvement. The evolutionary consequences of introgression have been addressed at the theoretical level, however, issues related to levels and patterns of introgression among (plant) species remain inadequately explored, including such factors as polyploidization, subgenome interaction inhabiting a common nucleus, and the genomic distribution and linkage relationships of introgressant alleles. We analyze introgression into the polyploid Gossypium hirsutum (upland cotton) from its sister G. tomentosum and compare the level and pattern with that of G. barbadense representing a different clade tracing to the same polyploidization. Across the genome, recurrent backcrossing to Gossypium hirsutum yielded only one-third of the expected average frequency of the G. tomentosum allele, although one unusual region showed preferential introgression. Although a similar rate of introgression is found in the two subgenomes of polyploid (AtDt) G. hirsutum, a preponderance of multilocus interactions were largely within the Dt subgenome. Skewed G. tomentosum chromatin transmission is polymorphic among two elite G. hirsutum genotypes, which suggests that genetic background may profoundly affect introgression of particular chromosomal regions. Only limited correspondence is found between G. hirsutum chromosomal regions that are intolerant to introgression from the two species, G. barbadense and G. tomentosum, concentrated near possible inversion polymorphisms. Complex transmission of introgressed chromatin highlights the challenges to utilization of exotic germplasm in crop improvement. © 2016

  9. Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.

    Directory of Open Access Journals (Sweden)

    Nina Winter

    Full Text Available BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

  10. Alternative epigenetic chromatin states of polycomb target genes.

    Directory of Open Access Journals (Sweden)

    Yuri B Schwartz

    2010-01-01

    Full Text Available Polycomb (PcG regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development.

  11. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    Science.gov (United States)

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P comet assay and the SCSA, which was not identified using traditional measures of sperm quality. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Directory of Open Access Journals (Sweden)

    Sha Ky

    2010-08-01

    Full Text Available Abstract Background Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i a source for purified cells (or nuclei of distinct types, and (ii a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models or to amalgamations of diverse cell types (tissue chunks, whole organisms. Results Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis, our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid genome. Conclusions Validating the DALEC mapping results, we observe a strong association

  13. Snake-like chromatin in conjunctival cells of normal elderly persons and of patients with primary Sjögren's syndrome and other connective tissue diseases

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1995-01-01

    Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell......Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell...

  14. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider 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 go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. 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 and then returned to its pre-damaged status to maintain epigenetic codes.

  15. Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization.

    Science.gov (United States)

    González-Romero, Rodrigo; Eirín-López, José M; Ausió, Juan

    2015-01-01

    High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance-most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Contribution of transposable elements and distal enhancers to evolution of human-specific features of interphase chromatin architecture in embryonic stem cells.

    Science.gov (United States)

    Glinsky, Gennadi V

    2018-03-01

    Transposable elements have made major evolutionary impacts on creation of primate-specific and human-specific genomic regulatory loci and species-specific genomic regulatory networks (GRNs). Molecular and genetic definitions of human-specific changes to GRNs contributing to development of unique to human phenotypes remain a highly significant challenge. Genome-wide proximity placement analysis of diverse families of human-specific genomic regulatory loci (HSGRL) identified topologically associating domains (TADs) that are significantly enriched for HSGRL and designated rapidly evolving in human TADs. Here, the analysis of HSGRL, hESC-enriched enhancers, super-enhancers (SEs), and specific sub-TAD structures termed super-enhancer domains (SEDs) has been performed. In the hESC genome, 331 of 504 (66%) of SED-harboring TADs contain HSGRL and 68% of SEDs co-localize with HSGRL, suggesting that emergence of HSGRL may have rewired SED-associated GRNs within specific TADs by inserting novel and/or erasing existing non-coding regulatory sequences. Consequently, markedly distinct features of the principal regulatory structures of interphase chromatin evolved in the hESC genome compared to mouse: the SED quantity is 3-fold higher and the median SED size is significantly larger. Concomitantly, the overall TAD quantity is increased by 42% while the median TAD size is significantly decreased (p = 9.11E-37) in the hESC genome. Present analyses illustrate a putative global role for transposable elements and HSGRL in shaping the human-specific features of the interphase chromatin organization and functions, which are facilitated by accelerated creation of novel transcription factor binding sites and new enhancers driven by targeted placement of HSGRL at defined genomic coordinates. A trend toward the convergence of TAD and SED architectures of interphase chromatin in the hESC genome may reflect changes of 3D-folding patterns of linear chromatin fibers designed to enhance both

  17. Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

    Science.gov (United States)

    Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

    2017-07-28

    Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

  18. Non coding RNA: sequence-specific guide for chromatin modification and DNA damage signaling

    Directory of Open Access Journals (Sweden)

    Sofia eFrancia

    2015-11-01

    Full Text Available 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 suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. 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.

  19. Large-scale Comparative Study of Hi-C-based Chromatin 3D Structure Modeling Methods

    KAUST Repository

    Wang, Cheng

    2018-05-17

    Chromatin is a complex polymer molecule in eukaryotic cells, primarily consisting of DNA and histones. Many works have shown that the 3D folding of chromatin structure plays an important role in DNA expression. The recently proposed Chro- mosome Conformation Capture technologies, especially the Hi-C assays, provide us an opportunity to study how the 3D structures of the chromatin are organized. Based on the data from Hi-C experiments, many chromatin 3D structure modeling methods have been proposed. However, there is limited ground truth to validate these methods and no robust chromatin structure alignment algorithms to evaluate the performance of these methods. In our work, we first made a thorough literature review of 25 publicly available population Hi-C-based chromatin 3D structure modeling methods. Furthermore, to evaluate and to compare the performance of these methods, we proposed a novel data simulation method, which combined the population Hi-C data and single-cell Hi-C data without ad hoc parameters. Also, we designed a global and a local alignment algorithms to measure the similarity between the templates and the chromatin struc- tures predicted by different modeling methods. Finally, the results from large-scale comparative tests indicated that our alignment algorithms significantly outperform the algorithms in literature.

  20. [Automated morphometric evaluation of the chromatin structure of liver cell nuclei after vagotomy].

    Science.gov (United States)

    Butusova, N N; Zhukotskiĭ, A V; Sherbo, I V; Gribkov, E N; Dubovaia, T K

    1989-05-01

    The morphometric analysis of the interphase chromatine structure of the hepatic cells nuclei was carried out on the automated TV installation for the quantitative analysis of images "IBAS-2" (by the OPTON firm, the FRG) according to 50 optical and geometric parameters during various periods (1.2 and 4 weeks) after the vagotomy operation. It is determined that upper-molecular organisation of chromatine undergoes the biggest changes one week after operation, and changes of granular component are more informative than changes of the nongranular component (with the difference 15-20%). It was also revealed that chromatine components differ in tinctorial properties, which are evidently dependent on physicochemical characteristics of the chromatine under various functional conditions of the cell. As a result of the correlation analysis the group of morphometric indices of chromatine structure was revealed, which are highly correlated with level of transcription activity of chromatine during various terms after denervation. The correlation quotient of these parameters is 0.85-0.97. The summing up: vagus denervation of the liver causes changes in the morphofunctional organisation of the chromatine.

  1. [Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

    Science.gov (United States)

    Fedina, A B; Gazarian, G G

    1976-01-01

    Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

  2. High-resolution mapping reveals links of HP1 with active and inactive chromatin components.

    Directory of Open Access Journals (Sweden)

    Elzo de Wit

    2007-03-01

    Full Text Available Heterochromatin protein 1 (HP1 is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5' regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2, which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5' ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components.

  3. A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

    Directory of Open Access Journals (Sweden)

    Julian Sosnik

    Full Text Available The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

  4. Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization

    International Nuclear Information System (INIS)

    Lebedev, D. V.; Filatov, M. V.; Kuklin, A. I.; Islamov, A. Kh.; Stellbrink, J.; Pantina, R. A.; Denisov, Yu. Yu.; Toperverg, B. P.; Isaev-Ivanov, V. V.

    2008-01-01

    The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 x 10 -1 to 10 -4 A -1 with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 μm and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure of the protein component of chromatin exhibits a fractal behavior on scales extending over two orders of magnitude, from the nucleosomal size to the size of an entire nucleus, and (3) the structure of the nucleic acid component of chromatin in chicken erythrocyte nuclei is likewise of a fractal nature and has two levels of organization or two phases with the crossover point at about 300-400 nm

  5. Effect of radiation and alkylating agents on chromatin degradation in normal and malignant lymphoid cells

    International Nuclear Information System (INIS)

    Ryabchenko, N.I.; Yurashkova, V.; Ivannik, B.P.; Konov, A.V.; Drashil, V.

    1991-01-01

    Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chormatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/Bl cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes

  6. The global relationship between chromatin physical topology, fractal structure, and gene expression

    DEFF Research Database (Denmark)

    Almassalha, Luay M; Tiwari, A; Ruhoff, P T

    2017-01-01

    in an empty space, but in a highly complex, interrelated, and dense nanoenvironment that profoundly influences chemical interactions. We explored the relationship between the physical nanoenvironment of chromatin and gene transcription in vitro. We analytically show that changes in the fractal dimension, D...... show that the increased heterogeneity of physical structure of chromatin due to increase in fractal dimension correlates with increased heterogeneity of gene networks. These findings indicate that the higher order folding of chromatin topology may act as a molecular-pathway independent code regulating...

  7. Vitamin D receptor (VDR) promoter targeting through a novel chromatin remodeling complex.

    Science.gov (United States)

    Kato, Shigeaki; Fujiki, Ryoji; Kitagawa, Hirochika

    2004-05-01

    We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.

  8. Extensive chromatin remodelling and establishment of transcription factor 'hotspots' during early adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; John, Sam

    2011-01-01

    hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides...... and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP-binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well-established role in activation of PPARγ...

  9. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

  10. Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Tibshirani, Michael; Zhao, Beibei; Gentil, Benoit J; Minotti, Sandra; Marques, Christine; Keith, Julia; Rogaeva, Ekaterina; Zinman, Lorne; Rouaux, Caroline; Robertson, Janice; Durham, Heather D

    2017-11-01

    Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS. © The Author 2017. Published by Oxford University Press. All rights reserved

  11. Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-beta binding in B cells.

    Science.gov (United States)

    Cruickshank, Mark N; Fenwick, Emily; Karimi, Mahdad; Abraham, Lawrence J; Ulgiati, Daniela

    2009-08-01

    Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

  12. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R.

    2005-01-01

    TIP48 is a highly conserved eukaryotic AAA + 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

  13. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  14. Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense.

    Science.gov (United States)

    Schillheim, Britta; Jansen, Irina; Baum, Stephani; Beesley, Alexander; Bolm, Carsten; Conrath, Uwe

    2018-03-01

    Modern crop production calls for agrochemicals that prime plants for enhanced defense. Reliable test systems for spotting priming-inducing chemistry, however, are rare. We developed an assay for the high-throughput search for compounds that prime microbial pattern-induced secretion of antimicrobial furanocoumarins (phytoalexins) in cultured parsley cells. The screen produced 1-isothiocyanato-4-methylsulfinylbutane (sulforaphane; SFN), a secondary metabolite in many crucifers, as a novel defense priming compound. While elucidating SFN's mode of action in defense priming, we found that in Arabidopsis ( Arabidopsis thaliana ) the isothiocyanate provokes covalent modification (K4me3, K9ac) of histone H3 in the promoter and promoter-proximal region of defense genes WRKY6 and PDF1 2 , but not PR1 SFN-triggered H3K4me3 and H3K9ac coincide with chromatin unpacking in the WRKY6 and PDF1 2 regulatory regions, primed WRKY6 expression, unprimed PDF1 2 activation, and reduced susceptibility to downy mildew disease ( Hyaloperonospora arabidopsidis ). Because SFN also directly inhibits H arabidopsidis and other plant pathogens, the isothiocyanate is promising for the development of a plant protectant with a dual mode of action. © 2018 American Society of Plant Biologists. All Rights Reserved.

  15. Adenovirus chromatin structure at different stages of infection

    Energy Technology Data Exchange (ETDEWEB)

    Daniell, E.; Groff, D.E.; Fedor, M.J.

    1981-12-01

    The authors investigated the structure of adenovirus deoxyribonecleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parental DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with (/sup 3/)thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure they identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. The authors found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

  16. ENCODE: A Sourcebook of Epigenomes and Chromatin Language

    Directory of Open Access Journals (Sweden)

    Maryam Yavartanoo

    2013-03-01

    Full Text Available Until recently, since the Human Genome Project, the general view has been that the majority of the human genome is composed of junk DNA and has little or no selective advantage to the organism. Now we know that this conclusion is an oversimplification. In April 2003, the National Human Genome Research Institute (NHGRI launched an international research consortium called Encyclopedia of DNA Elements (ENCODE to uncover non-coding functional elements in the human genome. The result of this project has identified a set of new DNA regulatory elements, based on novel relationships among chromatin accessibility, histone modifications, nucleosome positioning, DNA methylation, transcription, and the occupancy of sequence-specific factors. The project gives us new insights into the organization and regulation of the human genome and epigenome. Here, we sought to summarize particular aspects of the ENCODE project and highlight the features and data that have recently been released. At the end of this review, we have summarized a case study we conducted using the ENCODE epigenome data.

  17. Two independent modes of chromatin organization revealed by cohesin removal.

    Science.gov (United States)

    Schwarzer, Wibke; Abdennur, Nezar; Goloborodko, Anton; Pekowska, Aleksandra; Fudenberg, Geoffrey; Loe-Mie, Yann; Fonseca, Nuno A; Huber, Wolfgang; H Haering, Christian; Mirny, Leonid; Spitz, Francois

    2017-11-02

    Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-sized active and inactive compartments, and partitioning into sub-megabase domains (TADs). It remains unclear, however, how these layers of organization form, interact with one another and influence genome function. Here we show that deletion of the cohesin-loading factor Nipbl in mouse liver leads to a marked reorganization of chromosomal folding. TADs and associated Hi-C peaks vanish globally, even in the absence of transcriptional changes. By contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the three-dimensional organization of the genome results from the interplay of two independent mechanisms: cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; and cohesin-dependent formation of TADs, possibly by loop extrusion, which helps to guide distant enhancers to their target genes.

  18. Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.

    Science.gov (United States)

    Adachi, Akira; Senmatsu, Satoshi; Asada, Ryuta; Abe, Takuya; Hoffman, Charles S; Ohta, Kunihiro; Hirota, Kouji

    2018-05-03

    Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

  19. Epigenetic Alterations in Alzheimer’s Disease

    Science.gov (United States)

    Sanchez-Mut, Jose V.; Gräff, Johannes

    2015-01-01

    Alzheimer’s disease (AD) is the major cause of dementia in Western societies. It progresses asymptomatically during decades before being belatedly diagnosed when therapeutic strategies have become unviable. Although several genetic alterations have been associated with AD, the vast majority of AD cases do not show strong genetic underpinnings and are thus considered a consequence of non-genetic factors. Epigenetic mechanisms allow for the integration of long-lasting non-genetic inputs on specific genetic backgrounds, and recently, a growing number of epigenetic alterations in AD have been described. For instance, an accumulation of dysregulated epigenetic mechanisms in aging, the predominant risk factor of AD, might facilitate the onset of the disease. Likewise, mutations in several enzymes of the epigenetic machinery have been associated with neurodegenerative processes that are altered in AD such as impaired learning and memory formation. Genome-wide and locus-specific epigenetic alterations have also been reported, and several epigenetically dysregulated genes validated by independent groups. From these studies, a picture emerges of AD as being associated with DNA hypermethylation and histone deacetylation, suggesting a general repressed chromatin state and epigenetically reduced plasticity in AD. Here we review these recent findings and discuss several technical and methodological considerations that are imperative for their correct interpretation. We also pay particular focus on potential implementations and theoretical frameworks that we expect will help to better direct future studies aimed to unravel the epigenetic participation in AD. PMID:26734709

  20. Chromatin structure in the unicellular algae Olisthodiscus luteus, Crypthecodinium cohnii and Peridiniun balticum.

    Science.gov (United States)

    Rizzo, P J; Burghardt, R C

    1980-01-01

    Isolated nuclei of the unicellular alga Olisthodiscus luteus, the uninucleate dinoflagellate Crypthecodinium cohnii and the binucleate dinoflagellate Peridinium balticum were lysed and deposited on grids by the microcentrifugation technique. The ultrastructure of the released chromatin fibers was compared to that of mouse liver nuclei. Chromatin from nuclei of Olisthodiscus luteus and the "eukaryotic" nuclei of Peridinium balticum, appeared as linear arrays of regularly repeating subunits which were identical in size and morphology to mouse nucleosomes. In contrast, the chromatin fibers from Crypthecodinium cohnii nuclei appeared as smoothe threads with a diameter of about 6.5 nm. Nuclear preparations containing mixtures of "dinokaryotic" and "eukaryotic" nuclei of Peridinium balticum also contained smooth fibers which most likely originated from the dinokaryotic nuclei. These and other results demonstrating the presence of nucleosomes in lower eukaryotes suggest that the subunit structure of chromatin arose very early in the evolution of the eukaryotic cell.

  1. Macrogenomic engineering via modulation of the scaling of chromatin packing density.

    Science.gov (United States)

    Almassalha, Luay M; Bauer, Greta M; Wu, Wenli; Cherkezyan, Lusik; Zhang, Di; Kendra, Alexis; Gladstein, Scott; Chandler, John E; VanDerway, David; Seagle, Brandon-Luke L; Ugolkov, Andrey; Billadeau, Daniel D; O'Halloran, Thomas V; Mazar, Andrew P; Roy, Hemant K; Szleifer, Igal; Shahabi, Shohreh; Backman, Vadim

    2017-11-01

    Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

  2. Chromatin Structure in Bands and Interbands of Polytene Chromosomes Imaged by Atomic Force Microscopy

    NARCIS (Netherlands)

    de Grauw, C.J.; de Grauw, C.J.; Avogadro, A.; van den Heuvel, D.J.; van den Heuvel, D.J.; van der Werf, Kees; Otto, Cornelis; Kraan, Yvonne M.; van Hulst, N.F.; Greve, Jan

    1998-01-01

    Polytene chromosomes from Drosophila melanogaster, observed from squash preparations, and chromosomes from Chironomus thummi thummi, investigated under physiological conditions, are imaged using an Atomic Force Microscope. Various chromatin fiber structures can be observed with high detail in fixed

  3. Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

    Science.gov (United States)

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

    2014-09-25

    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.

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

    Directory of Open Access Journals (Sweden)

    Valeria Visone

    2014-09-01

    Full Text Available 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.

  5. Molecular composition of the W chromatin in some moth species studied by comparative genomic hybridization (CGH)

    Czech Academy of Sciences Publication Activity Database

    Sahara, K.; Marec, František; Eickhoff, U.; Traut, E.

    09, č. 1 (2001), s. 78 ISSN 0967-3849. [International Chromosome Conference /14./. 04.09.2001-08.09.2001, Wurzburg] Institutional research plan: CEZ:Av0Z5007907 Keywords : W chromatin Subject RIV: EB - Genetics ; Molecular Biology

  6. Applications of high-throughput sequencing to chromatin structure and function in mammals

    OpenAIRE

    Dunham, Ian

    2009-01-01

    High-throughput DNA sequencing approaches have enabled direct interrogation of chromatin samples from mammalian cells. We are beginning to develop a genome-wide description of nuclear function during development, but further data collection, refinement, and integration are needed.

  7. Arabidopsis Chromatin Assembly Factor 1 is required for occupancy and position of a subset of nucleosomes

    Czech Academy of Sciences Publication Activity Database

    Munoz-Viana, R.; Wildhaber, T.; Trejo-Arellano, M.S.; Mozgová, Iva; Hennig, L.

    2017-01-01

    Roč. 92, č. 3 (2017), s. 363-374 ISSN 0960-7412 Institutional support: RVO:61388971 Keywords : Arabidopsis thaliana * chromatin * CAF-1 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 5.901, year: 2016

  8. Introducing enteral feeding induces intestinal subclinical inflammation and respective chromatin changes in preterm pigs

    DEFF Research Database (Denmark)

    Willems, Rhea; Krych, Lukasz; Rybicki, Verena

    2015-01-01

    AIM: To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs. MATERIALS & METHODS: Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure......; no significant differences for colostrum) with corresponding decondensed chromatin configurations. On histology this correlated with mild mucosal lesions, particularly in formula-fed pigs. In CaCo-2 cells, histone hyperacetylation led to a marked increase in TLR4 mRNA and increased IL8 expression upon...... stimulation with lipopolysaccharide (median: 7.0; interquartile range: 5.63-8.85) compared with naive cells (median 4.2; interquartile range: 2.45-6.33; p = 0.03). CONCLUSION: Enteral feeding, particular with formula, induces subclinical inflammation in the premature intestine and more open chromatin...

  9. Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics

    Science.gov (United States)

    Aguilar, Carlos A.; Craighead, Harold G.

    2013-10-01

    Deoxyribonucleic acid (DNA) is the blueprint on which life is based and transmitted, but the way in which chromatin -- a dynamic complex of nucleic acids and proteins -- is packaged and behaves in the cellular nucleus has only begun to be investigated. Epigenetic modifications sit 'on top of' the genome and affect how DNA is compacted into chromatin and transcribed into ribonucleic acid (RNA). The packaging and modifications around the genome have been shown to exert significant influence on cellular behaviour and, in turn, human development and disease. However, conventional techniques for studying epigenetic or conformational modifications of chromosomes have inherent limitations and, therefore, new methods based on micro- and nanoscale devices have been sought. Here, we review the development of these devices and explore their use in the study of DNA modifications, chromatin modifications and higher-order chromatin structures.

  10. To spread or not to spread - chromatin modifications in response to DNA damage

    DEFF Research Database (Denmark)

    Altmeyer, M.; Lukas, J.

    2013-01-01

    Chromatin modifications in response to DNA damage are vital for genome integrity. Multiple proteins and pathways required to generate specialized chromatin domains around DNA lesions have been identified and the increasing amount of information calls for unifying concepts that would allow us...... to grasp the ever-increasing complexity. This review aims at contributing to this trend by focusing on feed-forward and feedback mechanisms, which in mammalian cells determine the extent of chromatin modifications after DNA damage. We highlight the emerging notion that the nodal points of these highly...... dynamic pathways operate in a rate-limiting mode, whose deregulation can disrupt physiological boundaries between damaged and undamaged chromatin, dictate repair pathway choice, and determine the fate of cells exposed to genotoxic stress....

  11. Local changes of higher-order chromatin structure during DSB-repair

    International Nuclear Information System (INIS)

    Falk, M; Lukasova, E; Gabrielova, B; Ondrej, V; Kozubek, S

    2008-01-01

    We show that double-strand breaks (DSBs) induced in DNA of human cells by γ-radiation arise mainly in active, gene-rich, decondensed chromatin. We demonstrate that DSBs show limited movement in living cells, occasionally resulting in their permanent clustering, which poses a risk of incorrect DNA rejoining. In addition, some DSBs remain unrepaired for several days after irradiation, forming lesions repairable only with difficulty which are hazardous for genome stability. These 'late' DSBs colocalize with heterochromatin markers (dimethylated histone H3 at lysine 9, HP1 and CENP-A proteins), despite the low density of the surrounding chromatin. This indicates that there is epigenetic silencing of loci close to unrepaired DSBs and/or stabilization of damaged decondensed chromatin loops during repair and post-repair reconstitution of chromatin structure

  12. Effect of Seminal Vesicles and Dithiotritol (Dtt on Stability of Sperm Chromatin

    Directory of Open Access Journals (Sweden)

    MH Nasr-Esfahani

    2005-04-01

    Full Text Available Introduction: Different studies have shown that there is no relation between sperm chromatin stability and fertilization rate in both IVF and ICSI patients. However, the relation between SDS tests, as a detergent, along with DTT as reducer of disulphide bridges has not been studied so far in ICSI patients. Since different concentrations of DTT can induce different degrees of sperm chromatin decondensation, the aim of this study was to evaluate the effect of different concentrations of DTT on sperm chromatin decondensation in IVF and ICSI cases. Methods: During this study, 85 patients were divided into two groups according to their treatment procedure (IVF or ICSI.Semen samples of each patient was evaluated for sperm chromatin tests including SDS, SDS+EDTA & SDS+DTT for assessment of free thiole groups level (-SH, amount of non covalent bond between Zn and thioles(-SH Zn SH- and levels of disulfide bond (-S-S- in sperm chromatin, respectively. In this study, seminal fructose concentration, corrected seminal fructose level and true corrected fructose level as indicators of seminal vesicle function on sperm chromatin stability were assessed. Results: No correlation was observed between any of the above tests and rate of fertilization, both in IVF and ICSI cases. However, in IVF patients, a significant correlation was observed between SDS, SDS+DTT test and seminal fructose level, while in ICSI patients, only a significant correlation was observed between SDS+DTT and corrected or true fructose concentration. Conclusion: Since no correlation was observed between sperm chromatin test and fertilization rate, it is suggested that the chromatin status of these samples are adequate for fertilization to take place and extent of disulphide bridges has no effect on fertilization rate. However, the amount of disulphide bound present in sperms of ICSI and IVF patients are different, and this difference is related to seminal vesicle performance in these patients.

  13. Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin

    OpenAIRE

    Freidkin, Ilya; Katcoff, Don J.

    2001-01-01

    In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1. In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1. The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype. In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is...

  14. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci

    DEFF Research Database (Denmark)

    Mousavi, Kambiz; Zare, Hossein; Dell'orso, Stefania

    2013-01-01

    )RNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding Pol II assembly. By nuclease sensitivity assay, we found that eRNAs regulate genomic access of the transcriptional...... complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events....

  15. ChromaSig: a probabilistic approach to finding common chromatin signatures in the human genome.

    Directory of Open Access Journals (Sweden)

    Gary Hon

    2008-10-01

    Full Text Available Computational methods to identify functional genomic elements using genetic information have been very successful in determining gene structure and in identifying a handful of cis-regulatory elements. But the vast majority of regulatory elements have yet to be discovered, and it has become increasingly apparent that their discovery will not come from using genetic information alone. Recently, high-throughput technologies have enabled the creation of information-rich epigenetic maps, most notably for histone modifications. However, tools that search for functional elements using this epigenetic information have been lacking. Here, we describe an unsupervised learning method called ChromaSig to find, in an unbiased fashion, commonly occurring chromatin signatures in both tiling microarray and sequencing data. Applying this algorithm to nine chromatin marks across a 1% sampling of the human genome in HeLa cells, we recover eight clusters of distinct chromatin signatures, five of which correspond to known patterns associated with transcriptional promoters and enhancers. Interestingly, we observe that the distinct chromatin signatures found at enhancers mark distinct functional classes of enhancers in terms of transcription factor and coactivator binding. In addition, we identify three clusters of novel chromatin signatures that contain evolutionarily conserved sequences and potential cis-regulatory elements. Applying ChromaSig to a panel of 21 chromatin marks mapped genomewide by ChIP-Seq reveals 16 classes of genomic elements marked by distinct chromatin signatures. Interestingly, four classes containing enrichment for repressive histone modifications appear to be locally heterochromatic sites and are enriched in quickly evolving regions of the genome. The utility of this approach in uncovering novel, functionally significant genomic elements will aid future efforts of genome annotation via chromatin modifications.

  16. A high-resolution map of the three-dimensional chromatin interactome in human cells.

    Science.gov (United States)

    Jin, Fulai; Li, Yan; Dixon, Jesse R; Selvaraj, Siddarth; Ye, Zhen; Lee, Ah Young; Yen, Chia-An; Schmitt, Anthony D; Espinoza, Celso A; Ren, Bing

    2013-11-14

    A large number of cis-regulatory sequences have been annotated in the human genome, but defining their target genes remains a challenge. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C)-based techniques. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C). We determined over one million long-range chromatin interactions at 5-10-kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts after TNF-α signalling in these cells. Unexpectedly, we found that TNF-α-responsive enhancers are already in contact with their target promoters before signalling. Such pre-existing chromatin looping, which also exists in other cell types with different extracellular signalling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is relatively stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.

  17. Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

    Science.gov (United States)

    Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

    2014-09-15

    Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM. © 2014. Published by The Company of Biologists Ltd.

  18. Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

    Science.gov (United States)

    Smith, Owen K.; Aladjem, Mirit I.

    2014-01-01

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

  19. Comparative analysis of chromatin landscape in regulatory regions of human housekeeping and tissue specific genes

    Directory of Open Access Journals (Sweden)

    Dasgupta Dipayan

    2005-05-01

    Full Text Available Abstract Background Global regulatory mechanisms involving chromatin assembly and remodelling in the promoter regions of genes is implicated in eukaryotic transcription control especially for genes subjected to spatial and temporal regulation. The potential to utilise global regulatory mechanisms for controlling gene expression might depend upon the architecture of the chromatin in and around the gene. In-silico analysis can yield important insights into this aspect, facilitating comparison of two or more classes of genes comprising of a large number of genes within each group. Results In the present study, we carried out a comparative analysis of chromatin characteristics in terms of the scaffold/matrix attachment regions, nucleosome formation potential and the occurrence of repetitive sequences, in the upstream regulatory regions of housekeeping and tissue specific genes. Our data show that putative scaffold/matrix attachment regions are more abundant and nucleosome formation potential is higher in the 5' regions of tissue specific genes as compared to the housekeeping genes. Conclusion The differences in the chromatin features between the two groups of genes indicate the involvement of chromatin organisation in the control of gene expression. The presence of global regulatory mechanisms mediated through chromatin organisation can decrease the burden of invoking gene specific regulators for maintenance of the active/silenced state of gene expression. This could partially explain the lower number of genes estimated in the human genome.

  20. A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

    Science.gov (United States)

    Singh, Hari R; Nardozza, Aurelio P; Möller, Ingvar R; Knobloch, Gunnar; Kistemaker, Hans A V; Hassler, Markus; Harrer, Nadine; Blessing, Charlotte; Eustermann, Sebastian; Kotthoff, Christiane; Huet, Sébastien; Mueller-Planitz, Felix; Filippov, Dmitri V; Timinszky, Gyula; Rand, Kasper D; Ladurner, Andreas G

    2017-12-07

    DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD + -metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Formation of DNA-protein crosslinks in gamma-irradiated chromatin

    International Nuclear Information System (INIS)

    Mee, L.K.

    1985-01-01

    Gamma-irradiation of chromatin in vitro and in vivo induces DNA-protein crosslinks which are stable to salt and detergent treatment. The efficiency of crosslink formation is 100 times greater in irradiated isolated chromatin than in chromatin irradiated in cells before isolation. Gamma-irradiation of isolated chromatin in the presence of radical scavengers shows that OH . is the most effective radical for the promotion of crosslinking whereas e/sub aq//sup -/ and O/sub 2//sup -/ are essentially ineffective. For chromatin irradiated in the cell before isolation, fewer crosslinks are formed in air than in an atmosphere of nitrogen; the greatest effect is found in cells irradiated in an atmosphere of nitrous oxide, suggesting that OH . may be involved in the formation of crosslinks in vivo. On the basis of comparing radiation-induced crosslinking in whole chromating (DNA, H1 histone, the core histones - H2A, H2B, H3 and H4 - and non-histone chromosomal proteins) and in a chromatin subunit (DNA and the core histones), the authors identified the core histones as the specific chromosomal proteins predominantly involved in crosslinking to DNA

  2. Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

    Directory of Open Access Journals (Sweden)

    Alexandre A.S.F. Raposo

    2015-03-01

    Full Text Available The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

  3. The impact of histones linked to sperm chromatin on embryo development and ART outcome.

    Science.gov (United States)

    Fournier, C; Labrune, E; Lornage, J; Soignon, G; Giscard d'Estaing, S; Guérin, J-F; Benchaib, M

    2018-03-02

    The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone-to-protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c-IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = -0.12, p ART procedure (c-IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR26%; p 26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy. © 2018 American Society of Andrology and European Academy of Andrology.

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

    Directory of Open Access Journals (Sweden)

    Sagar Darvekar

    Full Text Available 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.

  5. Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

    Directory of Open Access Journals (Sweden)

    Goran Lakisic

    2016-03-01

    Full Text Available BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi. In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

  6. Morphological changes of nuclear and chromatin architecture after microwave electromagnetic field exposure in 3T3 fibroblast cell cultures

    International Nuclear Information System (INIS)

    Mircea, D.; Chirila, Lavinia; Ciurea, A. V.; Helm, G.; Hankins, G.; Redrick, Jan; Gavrila, L.; Sheppard, B.; Bloodgoog, R.; Pallin, I.; Nitu, Rozalia; Rusu, I.

    2001-01-01

    It is already demonstrated in the literature that electromagnetic fields, particularly the microwave irradiation could be a powerful weapon against human tumors , but also against human body itself, depending on the wave parameters and irradiation time. The effects of microwave electromagnetic fields on living systems were studied in detail all over the world and, furthermore, the potential of intracellular damages by cytoskeleton, nuclear, chromatin and DNA alterations were carefully evaluated. In this study, the authors emphasize the morphological changes of nucleus and chromatin in fibroblast cell line 3T3 after microwave exposure with progressive increasing powers and times of irradiation. It was used a pulsed wave with 915 MHz frequency, with forward power ranging between 3 - 10 W, emitted by a helical microwave antenna placed into the cell culture medium, close to the cell monolayer. The authors tried to define certain severity stages of nuclear material alterations following different wave intensities and to compare these effects with other cytoplasmic organelle alterations. It was found that the nuclear material is the most sensitive intracellular structure in microwave electromagnetic field exposure. Also the authors tried to establish a well-defined protocol of irradiation with microwave electromagnetic fields in order to destroy the microtubule system of cytoskeleton in different types of cellular lines, in vitro. The cytoskeleton structure was evaluated by immunofluorescence methods. In non-muscle cells the cytoskeleton stability is achieved by interaction between microtubule system and actin filaments. Microtubule depolymerization by microwave exposure produces a secondary instability of cytoskeleton, the actin filaments coupling and cell contractility. The increasing of fibroblast contractility allows a more efficient treatment of the wounds with low spontaneous healing. Electromagnetic therapy could be an alternative therapy in plastic surgery

  7. Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

    2017-01-01

    The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

  8. Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

    Science.gov (United States)

    Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

    2013-01-30

    Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

    Science.gov (United States)

    Oliva, R; Mezquita, C

    1982-01-01

    In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA. Images PMID:7162988

  10. The possible role of chromatin conformation changes in adaptive responses to ionizing radiation

    International Nuclear Information System (INIS)

    Ekhtiar, A.; Ammer, A.; Jbawi, A.; Othman, A.

    2012-05-01

    Organisms are affected by different DNA damaging agents naturally present in the environment or released as a result of human activity. Many defense mechanisms have evolved in organisms to minimize genotoxic damage. One of them is induced radioresistance or adaptive response. The adaptive response could be considered as a nonspecific phenomenon in which exposure to minimal stress could result in increased resistance to higher levels of the same or to other types of stress some hours later. A better understanding of the molecular mechanism underlying the adaptive response may lead to an improvement of cancer treatment, risk assessment and risk management strategies, radiation protection. The aim of current study was to study the possible role of chromatin conformation changes induced by ionizing radiation on the adaptive responses in human lymphocyte. For this aim the chromatin conformation have been studied in human lymphocytes from three non-smoking and three smoking healthy volunteers prior, and after espouser to gamma radiation (adaptive dose 0.1 Gy, challenge dose 1.5 Gy and adaptive + dose challenge). Chromosomal aberrations and micronucleus have been used as end point to study radio cytotoxicity and adaptive response. Our results indicated individual differences in radio adaptive response and the level of this response was dependent of chromatin de condensation induced by a adaptive small dose.The results showed that different dose of gamma rays induce a chromatin de condensation in human lymphocyte. The maximum chromatin relaxation were record when lymphocyte exposed to adaptive dose (0.1 Gy.). Results also showed that Adaptive dose have affected on the induction of challenge dose (1.5 Gy) of chromosome aberration and micronucleus . The comparison of results of chromatin de condensation induction as measured by flow cytometry and cytogenetic damages measured by chromosomal aberrations or micronucleus, was showed a proportionality of adaptive response with

  11. APPLICATION OF THE SPERM CHROMATIN STRUCTURE ASSAY TO THE TEPLICE PROGRAM SEMEN STUDIES: A NEW METHOD FOR EVALUATING SPERM NUCLEAR CHROMATIN DAMAGE

    Science.gov (United States)

    ABSTRACTA measure of sperm chromatin integrity was added to the routine semen end points evaluated in the Teplice Program male reproductive health studies. To address the hypothesis that exposure to periods of elevated air pollution may be associated with abnormalities in sp...

  12. Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

    DEFF Research Database (Denmark)

    Fismen, S; Hedberg, A; Fenton, K A

    2009-01-01

    Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal i......Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo......-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic...... (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i...

  13. Rapid purification of recombinant histones.

    Science.gov (United States)

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  14. Systematic dissection of roles for chromatin regulators in a yeast stress response.

    Directory of Open Access Journals (Sweden)

    Assaf Weiner

    Full Text Available Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants and enzymes (chromatin modifier deletions we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.

  15. Relaxed selection against accidental binding of transcription factors with conserved chromatin contexts.

    Science.gov (United States)

    Babbitt, G A

    2010-10-15

    The spurious (or nonfunctional) binding of transcription factors (TF) to the wrong locations on DNA presents a formidable challenge to genomes given the relatively low ceiling for sequence complexity within the short lengths of most binding motifs. The high potential for the occurrence of random motifs and subsequent nonfunctional binding of many transcription factors should theoretically lead to natural selection against the occurrence of spurious motif throughout the genome. However, because of the active role that chromatin can influence over eukaryotic gene regulation, it may also be expected that many supposed spurious binding sites could escape purifying selection if (A) they simply occur in regions of high nucleosome occupancy or (B) their surrounding chromatin was dynamically involved in their identity and function. We compared nucleosome occupancy and the presence/absence of functionally conserved chromatin context to the strength of selection against spurious binding of various TF binding motifs in Saccharomyces yeast. While we find no direct relationship with nucleosome occupancy, we find strong evidence that transcription factors spatially associated with evolutionarily conserved chromatin states are under relaxed selection against accidental binding. Transcription factors (with/without) a conserved chromatin context were found to occur on average, (87.7%/49.3%) of their expected frequencies. Functional binding motifs with conserved chromatin contexts were also significantly shorter in length and more often clustered. These results indicate a role of chromatin context dependency in relaxing selection against spurious binding in nearly half of all TF binding motifs throughout the yeast genome. 2010 Elsevier B.V. All rights reserved.

  16. Relationship between chromatin complexity and nuclear envelope circularity in hippocampal pyramidal neurons

    International Nuclear Information System (INIS)

    Pantic, Igor; Basailovic, Milos; Paunovic, Jovana; Pantic, Senka

    2015-01-01

    Highlights: •We analyzed chromatin structure and nuclear envelope of 200 hippocampal pyramidal neurons. •Fractal and GLCM mathematical parameters were calculated each chromatin structure. •Nuclear shape was quantified by calculating circularity of the nuclear envelope. •Circularity was in significant relationship with chromatin fractal dimension. •Strong correlation was detected between circularity and some GLCM parameters. -- Abstract: In this study we tested the existence and strength of the relationship between circularity of nuclear envelope and mathematical parameters of chromatin structure. Coronal sections of the brain were made in 10 male albino mice. The brain tissue was stained using a modification of Feulgen method for DNA visualization. A total of 200 hippocampal pyramidal neurons (20 per animal) were visualized using DEM 200 High-Speed Color CMOS Chip and Olympus CX21FS1 microscope. Circularity of the nuclear membrane was calculated in ImageJ (NIH, USA) after the nuclear segmentation, based on the freehand selection of the nuclear regions of interest. Circularity was determined from the values of area and perimeter. For each chromatin structure, using fractal and grey level co-occurrence matrix (GLCM) algorithms, we determined the values of fractal dimension, lacunarity, angular second moment, GLCM entropy, inverse difference moment, GLCM correlation, and GLCM contrast. It was found that circularity is in a significant correlation (p < 0.05) with fractal dimension as the main parameter of fractal complexity analysis. Also, circularity was in a very strong relationship (p < 0.001) with certain parameters of grey level co-occurrence matrix such as the angular second moment and GLCM correlation. This is the first study to indicate that nuclear shape is significantly related to mathematical parameters of higher chromatin organization. Also, it seems that circularity of the nuclear envelope is a good predictor of certain features of chromatin

  17. Chromatin degradation under the effect of differentiation inductors and γ-radiation on thymus lymphocytes in vitro

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Sorokina, N.I.; Filippovich, I.V.

    1985-01-01

    Chemical inductors of differentiation were shown to cause chromatin degradation in thymus lymphocytes. This process was prevented by the protein synthesis inhibitors. The fragments formed after the effect of chemical differentiation inductors on thymocytes were fully identical to chromatin internucleosome degradation products formed in the exposed cells. Chromatin degradation under the effect of chemical differentiation inductors was most pronounced in a more radiosensitive thymocyte fraction

  18. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

    Directory of Open Access Journals (Sweden)

    Varvara A Khoroshko

    Full Text Available Late-replicating domains (intercalary heterochromatin in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW, and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a

  19. Structural Modeling of GR Interactions with the SWI/SNF Chromatin Remodeling Complex and C/EBP

    DEFF Research Database (Denmark)

    Muratcioglu, Serena; Presman, Diego M; Pooley, John R

    2015-01-01

    The glucocorticoid receptor (GR) is a steroid-hormone-activated transcription factor that modulates gene expression. Transcriptional regulation by the GR requires dynamic receptor binding to specific target sites located across the genome. This binding remodels the chromatin structure to allow...... interaction with other transcription factors. Thus, chromatin remodeling is an essential component of GR-mediated transcriptional regulation, and understanding the interactions between these molecules at the structural level provides insights into the mechanisms of how GR and chromatin remodeling cooperate...

  20. Studies on the Chromatin Isolated from the Organs of Animals Received Whole-body X-ray Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Han, Su Nam [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1967-09-15

    Within experimental chromatin, the total protein: DNA ratio did not vary in the same organs of control and irradiated rats. However, the amount of RNA and total protein associated with the DNA varied considerably among the different types of chromatin. In particular, the content of chromatin was the highest in the irradiated tissue, and the lowest in the chromatin control tissue. RNA and total protein ratio of chromatins from brain, liver, testis and spleen declined with experimental organs. 2) There was the same quantitative relationship between the amount of RNA and the amount histone-protein associated with DNA in each chromatin. 3) RNA:DNA ratio of chromatin showed a 1.5-2 times increase in the irradiated organs except brain. However, RNA:DNA ratio was decreased in chromatin by irradiation. 4) Histone-protein: Residual protein ratio was greatly varied among the organs. However, the effect was not found by irradiation. 5) Priming activity of chromatins showed a higher value in testis and the activity was greater in organs with higher metabolic activity. 6) Inhibition of Actinomycin D observable in chromatin for testis, liver, spleen and brain declined without relationship between irradiated and non-irradiated conditions. Ammonium sulfate in DNA of chromatin from histone showed increased priming activity with dissociation by Electrostatics. It may give different effect of ammonium sulfate on stimulation by property of chromatins. 7) It is suggested that the results support a proposal that the higher sensitivity of radioactive in testis, spleen by irradiated showed a increase and decrease lower-sensitivity of radioactive from brain, liver than did priming activity under the radioactive conditions.

  1. Electron microscopic study on the initial effect of gamma-irradiation on the chromatin structure of L cells

    International Nuclear Information System (INIS)

    Kondo, Takashi; Nakanishi, Y.H.; Yoshii, Giichi

    1979-01-01

    Mouse L cells are gamma-irradiated at a dose of 1 Mrad, and ultrathin sections of the cells are examined by electron microscopy. The distance between chromatin fibers in diffused chromatin regions in the irradiated nuclei is essentially identical with the nonirradiated control. In contrast, an increase of the distance between the chromatin fibers is observed in the excess of Ca ions in irradiation. (author)

  2. Disruption of the Interaction of the Androgen Receptor with Chromatin: A Novel Therapeutic Approach in Prostate Cancer

    Science.gov (United States)

    2016-10-01

    AWARD NUMBER: W81XWH-15-1-0543 TITLE: Disruption of the Interaction of the Androgen Receptor with Chromatin : A Novel Therapeutic Approach in...DATES COVERED 8 Sep 2015 - 7 Sep 2016 4. TITLE AND SUBTITLE Disruption of the Interaction of the Androgen Receptor with Chromatin : A Novel 5a. CONTRACT...1: Select and evaluate peptides/peptidomimetics in models of PCa. Aim 2: Determine the molecular action of peptide /peptidomimetics at the chromatin

  3. Studies on the Chromatin Isolated from the Organs of Animals Received Whole-body X-ray Irradiation

    International Nuclear Information System (INIS)

    Han, Su Nam

    1967-01-01

    Within experimental chromatin, the total protein: DNA ratio did not vary in the same organs of control and irradiated rats. However, the amount of RNA and total protein associated with the DNA varied considerably among the different types of chromatin. In particular, the content of chromatin was the highest in the irradiated tissue, and the lowest in the chromatin control tissue. RNA and total protein ratio of chromatins from brain, liver, testis and spleen declined with experimental organs. 2) There was the same quantitative relationship between the amount of RNA and the amount histone-protein associated with DNA in each chromatin. 3) RNA:DNA ratio of chromatin showed a 1.5-2 times increase in the irradiated organs except brain. However, RNA:DNA ratio was decreased in chromatin by irradiation. 4) Histone-protein: Residual protein ratio was greatly varied among the organs. However, the effect was not found by irradiation. 5) Priming activity of chromatins showed a higher value in testis and the activity was greater in organs with higher metabolic activity. 6) Inhibition of Actinomycin D observable in chromatin for testis, liver, spleen and brain declined without relationship between irradiated and non-irradiated conditions. Ammonium sulfate in DNA of chromatin from histone showed increased priming activity with dissociation by Electrostatics. It may give different effect of ammonium sulfate on stimulation by property of chromatins. 7) It is suggested that the results support a proposal that the higher sensitivity of radioactive in testis, spleen by irradiated showed a increase and decrease lower-sensitivity of radioactive from brain, liver than did priming activity under the radioactive conditions.

  4. Effect of benzimidazol-derivatives on the DNA-protein binding formation after UV-radiation of chromatin

    International Nuclear Information System (INIS)

    Mil', E.M.; Binyukov, V.I.; Zhil'tsova, V.M.; Stolyarova, L.G.; Kuznetsov, Yu.V.

    1991-01-01

    Effect of benzimidazol-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazol interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA, whilt aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1

  5. Parvovirus induced alterations in nuclear architecture and dynamics.

    Directory of Open Access Journals (Sweden)

    Teemu O Ihalainen

    2009-06-01

    Full Text Available The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analysis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.

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

    Science.gov (United States)

    Wedemann, Gero; Langowski, Jörg

    2002-06-01

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

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

    International Nuclear Information System (INIS)

    Öberg, Christine; Belikov, Sergey

    2012-01-01

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

  8. Radiation-induced thymine base damage and its excision repair in active and inactive chromatin of HeLa cells

    International Nuclear Information System (INIS)

    Patil, M.S.; Locher, S.E.; Hariharan, P.V.

    1985-01-01

    The extent of production and excision repair of 5,6-dihydroxydihydrothymine type base (t') damage was determined in transcriptionally active and inactive chromatin of HeLa cells after exposure to 6.8 MeV electrons. It was observed that not only the yield but also rate of repair of t' products was greater in the active chromatin compared to the inactive chromatin of HeLa cells. The results strongly indicate that the conformation of chromatin is an important factor in determining the sensitivity to radiation damage and accessibility to enzymes required for repair of such damage. (author)

  9. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements

    DEFF Research Database (Denmark)

    Grøntved, Lars; John, Sam; Baek, Songjoon

    2013-01-01

    -binding sites are occupied by C/EBPβ. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPβ for GR recruitment. Disruption of C/EBPβ binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin...... remodelling specifically at sites co-occupied by GR and C/EBPβ. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPβ regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell...

  10. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    Science.gov (United States)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  11. Fragmentation of chromatin DNA in mouse thymus cells after whole body γ-irradiation

    International Nuclear Information System (INIS)

    Wei Kang; Liu Xueying; Zhu Xuefen

    1984-01-01

    The characteristics of soluble chromatin in mouse thymus nuclei after whole body γ-irradiation were investigated by means of polyacrylamide gel electrophoresis. After deproteinization and electrophoresis eight regular DNA bands were revealed. The molecular weights of these bands were estimated by comparing their migration rates with those of the standard fragments obtained from PBR 322 digested completely by restrictive endonuclease Hae III. The molecular weight of the first band was calculated to be 186 base pairs corresponding approximately to the size of DNA fragment from a single nucleosome, and those of other bands appeared to be its multiples. The results suggested that the disintegration of chromatin DNA after γ-irradiation might have occurred at the linkage regions of chromatin. The autolysis product of normal thymus chromatin under sterile condition were also analyzed and its electrophoretic pattern was found to be just the same as that of the postirradiation product. It seems, therefore, that the endonuclease existing in normal tissues might be responsible for the postirradiation chromatin degradation. The mechanism of this kind of enzymatic digestion remains to be elucidated in further investigation. (author)

  12. EBNA1 efficiently assembles on chromatin containing the Epstein-Barr virus latent origin of replication

    International Nuclear Information System (INIS)

    Avolio-Hunter, Tina M.; Frappier, Lori

    2003-01-01

    The Epstein-Barr virus (EBV) protein, EBNA1, activates the replication of latent EBV episomes and the transcription of EBV latency genes by binding to recognition sites in the DS and FR elements of oriP. Since EBV episomes exist as chromatin, we have examined the interaction of EBNA1 with oriP templates assembled with physiologically spaced nucleosomes. We show that EBNA1 retains the ability to efficiently bind its recognition sites within the DS and FR elements in oriP chromatin and that this property is intrinsic to the EBNA1 DNA binding domain. The efficient assembly of EBNA1 on oriP chromatin does not require ATP-dependent chromatin remodeling factors and does not cause the precise positioning of nucleosomes within or adjacent to the FR and DS elements. Thus EBNA1 belongs to a select group of proteins that can efficiently access their recognition sites within nucleosomes without the need for additional chromatin remodeling factors

  13. Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

    Science.gov (United States)

    Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

    2017-01-01

    Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

  14. Mass Spectrometry-Based Proteomics for the Analysis of Chromatin Structure and Dynamics

    Directory of Open Access Journals (Sweden)

    Monica Soldi

    2013-03-01

    Full Text Available Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs and DNA methylation, which act in a concerted manner to enforce a specific “chromatin landscape”, with a regulatory effect on gene expression. Mass Spectrometry (MS has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from “Bottom Up” to “Top Down” analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays.

  15. Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

    Science.gov (United States)

    Leonova, Olga G; Karajan, Bella P; Ivlev, Yuri F; Ivanova, Julia L; Skarlato, Sergei O; Popenko, Vladimir I

    2013-01-01

    We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

  16. ERECTA signaling controls Arabidopsis inflorescence architecture through chromatin-mediated activation of PRE1 expression.

    Science.gov (United States)

    Cai, Hanyang; Zhao, Lihua; Wang, Lulu; Zhang, Man; Su, Zhenxia; Cheng, Yan; Zhao, Heming; Qin, Yuan

    2017-06-01

    Flowering plants display a remarkable diversity in inflorescence architecture, and pedicel length is one of the key contributors to this diversity. In Arabidopsis thaliana, the receptor-like kinase ERECTA (ER) mediated signaling pathway plays important roles in regulating inflorescence architecture by promoting cell proliferation. However, the regulating mechanism remains elusive in the pedicel. Genetic interactions between ERECTA signaling and the chromatin remodeling complex SWR1 in the control of inflorescence architecture were studied. Comparative transcriptome analysis was applied to identify downstream components. Chromatin immunoprecipitation and nucleosome occupancy was further investigated. The results indicated that the chromatin remodeler SWR1 coordinates with ERECTA signaling in regulating inflorescence architecture by activating the expression of PRE1 family genes and promoting pedicel elongation. It was found that SWR1 is required for the incorporation of the H2A.Z histone variant into nucleosomes of the whole PRE1 gene family and the ERECTA controlled expression of PRE1 gene family through regulating nucleosome dynamics. We propose that utilization of a chromatin remodeling complex to regulate gene expression is a common theme in developmental control across kingdoms. These findings shed light on the mechanisms through which chromatin remodelers orchestrate complex transcriptional regulation of gene expression in coordination with a developmental cue. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  17. Evaluation of chromatin integrity of motile bovine spermatozoa capacitated in vitro.

    Science.gov (United States)

    Reckova, Z; Machatkova, M; Rybar, R; Horakova, J; Hulinska, P; Machal, L

    2008-08-01

    The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF-TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n=3, n=5 and n=9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

  18. Nuclear and chromatin structures and their influence on the radiosensitivity of DNA

    International Nuclear Information System (INIS)

    Oleinick, N.L.; Chiu, S.-M.

    1994-01-01

    Among the factors contributing to the distribution of DNA damage within irradiated mammalian cell nuclei are the interactions of DNA with nuclear proteins and the formation of multi-molecular chromatin structures. Studies on the manipulation of chromatin structures of isolated nuclei are summarised. The majority of chromatin within the nucleus of living cells is tightly compacted into nucleosomal superhelices and other higher order structures which have a limited ability to be damaged by radiation. The treatment of isolated nuclei with hypotonic buffers causes a decondensation of these structures and markedly sensitises the DNA to radiation, while retaining the majority of the chromosomal proteins. On the other hand, treatment of nuclei with hypertonic buffers strips the DNA of specific classes of nuclear proteins, destroying chromatin structure, and this procedure also enhances the sensitivity of the DNA to radiation. The various expanded chromatin structures are models for the structure of the minor fraction of DNA which is decondensed in preparation for transcription or replication. The combined results indicate that the majority of nuclear DNA is protected by histones and other nuclear proteins from radiation damage, partially as a result of the limited accessibility of the condensed structures to hydroxyl radical and partially as a result of the scavenging of radicals by the proteins. (Author)

  19. Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

    Science.gov (United States)

    Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

    2003-01-01

    The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

  20. Effect of ultraviolet irradiation on chromatin and its components from Yoshida ascites tumour cells

    International Nuclear Information System (INIS)

    Ramakrishnan, N.; Patil, M.S.; Pradhan, D.S.

    1981-01-01

    A study has been made of the effect of U.V. irradiation on Yoshida ascites tumour chromatin and its non-DNA components. The extractability of total histones was increased from 6% to 17% with an increase in U.V. incident radiation dose from 500J/m 2 to 2000J/m 2 . The polyacrylamide gel electrophoresis pattern of chromosomal proteins was examined after irradiation of the chromatin, and the effect of U.V. irradiation of chromatin on histones was also investigated. The results indicated that cross-linking of DNA with chromosomal proteins is an important category of U.V. radiation-induced lesions discerned in U.V. irradiated chromatin. Histones and several non-histone proteins seemed to undergo U.V. radiation-induced cross-linking with DNA, which was taken as indicative of their close association with DNA in the chromatin structure. It is suggested that the cross-link formation between DNA and non-histone proteins may be due to sequence-specific association of non-histone proteins with DNA. (U.K.)

  1. The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.

    Science.gov (United States)

    Daer, René M; Cutts, Josh P; Brafman, David A; Haynes, Karmella A

    2017-03-17

    In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

  2. Saccharomyces cerevisiae Linker Histone—Hho1p Maintains Chromatin Loop Organization during Ageing

    Directory of Open Access Journals (Sweden)

    Katya Uzunova

    2013-01-01

    Full Text Available Intricate, dynamic, and absolutely unavoidable ageing affects cells and organisms through their entire lifetime. Driven by diverse mechanisms all leading to compromised cellular functions and finally to death, this process is a challenge for researchers. The molecular mechanisms, the general rules that it follows, and the complex interplay at a molecular and cellular level are yet little understood. Here, we present our results showing a connection between the linker histones, the higher-order chromatin structures, and the process of chronological lifespan of yeast cells. By deleting the gene for the linker histone in Saccharomyces cerevisiae we have created a model for studying the role of chromatin structures mainly at its most elusive and so far barely understood higher-order levels of compaction in the processes of yeast chronological lifespan. The mutant cells demonstrated controversial features showing slower growth than the wild type combined with better survival during the whole process. The analysis of the global chromatin organization during different time points demonstrated certain loss of the upper levels of chromatin compaction in the cells without linker histone. The results underlay the importance of this histone for the maintenance of the chromatin loop structures during ageing.

  3. Regulation of chromatin states by drugs of abuse.

    Science.gov (United States)

    Walker, Deena M; Cates, Hannah M; Heller, Elizabeth A; Nestler, Eric J

    2015-02-01

    Drug addiction involves long-term behavioral abnormalities and gene expression changes throughout the mesolimbic dopamine system. Epigenetic mechanisms establish/maintain alterations in gene expression in the brain, providing the impetus for investigations characterizing how epigenetic processes mediate the effects of drugs of abuse. This review focuses on evidence that epigenetic events, specifically histone modifications, regulate gene expression changes throughout the reward circuitry. Drugs of abuse induce changes in histone modifications throughout the reward circuitry by altering histone-modifying enzymes, manipulation of which reveals a role for histone modification in addiction-related behaviors. There is a complex interplay between these enzymes, resulting in a histone signature of the addicted phenotype. Insights gained from these studies are key to identifying novel targets for diagnosis and therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Concerted Flexibility of Chromatin Structure, Methylome, and Histone Modifications along with Plant Stress Responses

    Directory of Open Access Journals (Sweden)

    Ana Paula Santos

    2017-01-01

    Full Text Available The spatial organization of chromosome structure within the interphase nucleus, as well as the patterns of methylome and histone modifications, represent intersecting layers that influence genome accessibility and function. This review is focused on the plastic nature of chromatin structure and epigenetic marks in association to stress situations. The use of chemical compounds (epigenetic drugs or T-DNA-mediated mutagenesis affecting epigenetic regulators (epi-mutants are discussed as being important tools for studying the impact of deregulated epigenetic backgrounds on gene function and phenotype. The inheritability of epigenetic marks and chromatin configurations along successive generations are interpreted as a way for plants to “communicate” past experiences of stress sensing. A mechanistic understanding of chromatin and epigenetics plasticity in plant response to stress, including tissue- and genotype-specific epigenetic patterns, may help to reveal the epigenetics contributions for genome and phenotype regulation.

  5. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

  6. FIND: difFerential chromatin INteractions Detection using a spatial Poisson process.

    Science.gov (United States)

    Djekidel, Mohamed Nadhir; Chen, Yang; Zhang, Michael Q

    2018-02-12

    Polymer-based simulations and experimental studies indicate the existence of a spatial dependency between the adjacent DNA fibers involved in the formation of chromatin loops. However, the existing strategies for detecting differential chromatin interactions assume that the interacting segments are spatially independent from the other segments nearby. To resolve this issue, we developed a new computational method, FIND, which considers the local spatial dependency between interacting loci. FIND uses a spatial Poisson process to detect differential chromatin interactions that show a significant difference in their interaction frequency and the interaction frequency of their neighbors. Simulation and biological data analysis show that FIND outperforms the widely used count-based methods and has a better signal-to-noise ratio. © 2018 Djekidel et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Binding of polycyclic and nitropolycyclic aromatic hydrocarbons to specific fractions of rat lung chromatin

    International Nuclear Information System (INIS)

    Mitchell, C.E.; Akkaraju, S.

    1988-01-01

    Binding of polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons (NPAH) to rat lung nuclei was investigated. Following carcinogen exposure, nuclei were fractionated into active chromatin, nuclear matrix, low salt, and high salt fractions. Preferential binding to active chromatin and nuclear matrix fractions was observed for benzo(a)pyrene (BP), 6-nitro benzo(a)pyrene, 1,6-dinitropyrene (1,6-DNP), and 1-nitropyrene. Incubation of nuclei with BP, benzo(a)pyrene diolepoxide (BPDE), and 1,6-DNP showed that the selective binding was dependent upon the concentration of chemical with less selectivity at higher concentrations. This study shows that NPAH should be considered as another class of compounds that may exert their biological effects by binding to selected regions of chromatin that are involved in DNA replication and translation. (author)

  8. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    International Nuclear Information System (INIS)

    Teif, Vladimir B; Rippe, Karsten

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

  9. Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Filippovich, I.V.

    1986-01-01

    A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

  10. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    International Nuclear Information System (INIS)

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-01-01

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ

  11. Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection.

    Science.gov (United States)

    Kulej, Katarzyna; Avgousti, Daphne C; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N; Kim, Eui Tae; Garcia, Benjamin A; Weitzman, Matthew D

    2017-04-01

    Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  13. Radiation-induced XRCC4 association with chromatin DNA analyzed by biochemical fractionation

    International Nuclear Information System (INIS)

    Kamdar, R.P.; Matsumoto, Yoshihisa

    2010-01-01

    XRCC4, in association with DNA ligase IV, is thought to play a critical role in the ligation of two DNA ends in DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ) pathway. In the present study, we captured radiation-induced chromatin-recruitment of XRCC4 by biochemical fractionation using detergent Nonidet P-40. A subpopulation of XRCC4 changed into a form that is resistant to the extraction with 0.5% Nonidet P-40-containing buffer after irradiation. This form of XRCC4 was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA. This chromatin-recruitment of XRCC4 could be seen immediately (<0.1 hr) after irradiation and remained up to 4 hr after 20 Gy irradiation. It was seen even after irradiation of small doses, id est (i.e.), 2 Gy, but the residence of XRCC4 on chromatin was very transient after 2 Gy irradiation, returning to near normal level in 0.2-0.5 hr after irradiation. The chromatin-bound XRCC4 represented only -1% of total XRCC4 molecules even after 20 Gy irradiation and the quantitative analysis using purified protein as the reference suggested that only a few XRCC4-DNA ligase IV complexes were recruited to each DNA end. We further show that the chromatin-recruitment of XRCC4 was not attenuated by wortmannin, an inhibitor of DNA-PK, or siRNA-mediated knockdown of the DNA-PK catalytic subunit (DNA-PKcs), indicating that this process does not require DNA-PKcs. These results would provide us with useful experimental tools and important insights to understand the DNA repair process through NHEJ pathway. (author)

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

    Science.gov (United States)

    Olson, Wilma

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

  15. Genetics, chromatin diminution, and sex chromosome evolution in the parasitic nematode genus Strongyloides.

    Science.gov (United States)

    Nemetschke, Linda; Eberhardt, Alexander G; Hertzberg, Hubertus; Streit, Adrian

    2010-10-12

    When chromatin diminution occurs during a cell division a portion of the chromatin is eliminated, resulting in daughter cells with a smaller amount of genetic material. In the parasitic roundworms Ascaris and Parascaris, chromatin diminution creates a genetic difference between the soma and the germline. However, the function of chromatin diminution remains a mystery, because the vast majority of the eliminated DNA is noncoding. Within the parasitic roundworm genus Strongyloides, S. stercoralis (in man) and S. ratti (in rat) employ XX/XO sex determination, but the situation in S. papillosus (in sheep) is different but controversial. We demonstrate genetically that S. papillosus employs sex-specific chromatin diminution to eliminate an internal portion of one of the two homologs of one chromosome pair in males. Contrary to ascarids, the eliminated DNA in S. papillosus contains a large number of genes. We demonstrate that the region undergoing diminution is homologous to the X chromosome of the closely related S. ratti. The flanking regions, which are not diminished, are homologous to the S. ratti autosome number I. Furthermore, we found that the diminished chromosome is not incorporated into sperm, resulting in a male-specific transmission ratio distortion. Our data indicate that on the evolutionary path to S. papillosus, the X chromosome fused with an autosome. Chromatin diminution serves to functionally restore an XX/XO sex-determining system. A consequence of the fusion and the process that copes with it is a transmission ratio distortion in males for certain loci. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. 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.

    Science.gov (United States)

    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

  17. Possible nature and specificity of a protein factor favoringsolubilization of chromatin from irradiated animal thymocytes

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Trebenok, Z.A.; Filippovich, I.V.

    1989-01-01

    It is shown that activation of endonucleolysis of thymocyte nuclear chromatin by protein factor from the cells of irradiated animals is not conditioned by its nuclease activity or ability to activate Ca 2+ , Mg 2+ - dependent lymphocyte endonuclease. DNA degradation character and kinetics of accumulation of the forming products doesn't change in autolysis of thymocyte nucleus. It is assumed that protein factor doesn't participate in starting mechanisms of postirradiation chromatin degradation but can be of significance at delayed stages of the process. The discovered effect is characterized by tissue and specific characteristic

  18. Protocol: methodology for chromatin immunoprecipitation (ChIP in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Strenkert Daniela

    2011-11-01

    Full Text Available Abstract We report on a detailed chromatin immunoprecipitation (ChIP protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.

  19. Chromatin Modifying Agents in the In Vitro Production of Bovine Embryos

    Directory of Open Access Journals (Sweden)

    Fabio Morato Monteiro

    2011-01-01

    Full Text Available The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species.

  20. Gametophyte differentiation and imprinting control in plants: Crosstalk between RBR and chromatin.

    Science.gov (United States)

    Johnston, Amal J; Gruissem, Wilhelm

    2009-01-01

    The Retinoblastoma (pRb) pathway has been implicated as a convergent regulatory unit in the control of cell cycle and disease. We have shown that a crosstalk between RETINOBLASTOMA RELATED (RBR), the Arabidopsis homologue of pRb, and the genes encoding proteins of the chromatin complexes involved in DNA or histone methylation, controls gametophytic and post-fertilization differentiation events and a subset of imprinting effects. We describe here a plausible model that incorporates several components of the plant Retinoblastoma pathway, thus offering a novel paradigm that merges the traditional cell cycle and the chromatin components in the control of cell differentiation and imprinting.