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Sample records for abnormal chromatin condensation

  1. CHD5 is required for spermiogenesis and chromatin condensation.

    PubMed

    Zhuang, Tiangang; Hess, Rex A; Kolla, Venkatadri; Higashi, Mayumi; Raabe, Tobias D; Brodeur, Garrett M

    2014-02-01

    Haploid spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa during spermiogenesis. Abnormalities in these steps can lead to serious male fertility problems, from oligospermia to complete azoospermia. CHD5 is a chromatin-remodeling nuclear protein expressed almost exclusively in the brain and testis. Male Chd5 knockout (KO) mice have deregulated spermatogenesis, characterized by immature sloughing of spermatids, spermiation failure, disorganization of the spermatogenic cycle and abnormal head morphology in elongating spermatids. This results in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Sperm that did enter the epididymis displayed irregular shaped sperm heads, and retained cytoplasmic components. These sperm also stained positively for acidic aniline, indicating improper removal of histones and lack of proper chromatin condensation. Electron microscopy showed that spermatids in the seminiferous tubules of Chd5 KO mice have extensive nuclear deformation, with irregular shaped heads of elongated spermatids, and lack the progression of chromatin condensation in an anterior-to-posterior direction. However, the mRNA expression levels of other important genes controlling spermatogenesis were not affected. Chd5 KO mice also showed decreased H4 hyperacetylation beginning at stage IX, step 9, which is vital for the histone-transition protein replacement in spermiogenesis. Our data indicate that CHD5 is required for normal spermiogenesis, especially for spermatid chromatin condensation.

  2. Quantification of chromatin condensation level by image processing.

    PubMed

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

    2014-03-01

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

  3. Premature chromatin condensation upon accumulation of NIMA.

    PubMed Central

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

    1994-01-01

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

  4. Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques.

    PubMed

    Irez, T; Sahmay, S; Ocal, P; Goymen, A; Senol, H; Erol, N; Kaleli, S; Guralp, O

    2015-05-01

    The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)-EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS-EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy-positive group compared to the negative group, both in IUI (P = 0.042) and ICSI groups (P = 0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P = 0.015, r = 0.214 and P = 0.014, r = 0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.

  5. A NIMA homologue promotes chromatin condensation in fission yeast.

    PubMed

    Krien, M J; Bugg, S J; Palatsides, M; Asouline, G; Morimyo, M; O'Connell, M J

    1998-04-01

    Entry into mitosis requires p34(cdc2), which activates downstream mitotic events through phosphorylation of key target proteins. In Aspergillus nidulans, the NIMA protein kinase has been identified as a potential downstream target and plays a role in regulating chromatin condensation at mitosis. nimA- mutants arrest in a state that physically resembles interphase even though p34(cdc2) is fully active. Despite evidence for the existence of NIMA-like activities in a variety of cell types, the only bona fide NIMA homologue that has been identified is the nim-1 gene of Neurospora crassa. We report here the isolation of a fission yeast NIMA homologue, and have designated this gene fin1 and the 83 kDa predicted protein p83(fin1). Overexpression of fin1 promotes premature chromatin condensation from any point in the cell cycle independently of p34(cdc2) function. Like NIMA, p83(fin1) levels fluctuate through the cell cycle, peaking in mitosis and levels are greatly elevated by removal of C-terminal PEST sequences. Deletion of fin1 results in viable but elongated cells, indicative of a cell cycle delay. Genetic analysis has placed this delay in G2 but, unlike in nimA mutants of Aspergillus, p34(cdc2) activation appears to be delayed. Interaction of fin1 mutants with other strains defective in chromatin organisation also support the hypothesis of p83(fin1) playing a role in this process at the onset of mitosis. These data indicate that NIMA-related kinases may be a general feature of the cell cycle and chromatin organisation at mitosis.

  6. Formation of mammalian erythrocytes: chromatin condensation and enucleation.

    PubMed

    Ji, Peng; Murata-Hori, Maki; Lodish, Harvey F

    2011-07-01

    In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast chromatin condensation and enucleation, and conclude with our perspectives on future studies.

  7. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

    SciTech Connect

    Finan, John D.; Leddy, Holly A.; Guilak, Farshid

    2011-05-06

    Highlights: {yields} The rate of nucleocytoplasmic transport increases under hyper-osmotic stress. {yields} The mechanism is a change in nuclear geometry, not a change in permeability of the nuclear envelope. {yields} Intracytoplasmic but not intranuclear diffusion is sensitive to osmotic stress. {yields} Pores in the chromatin of the nucleus enlarge under hyper-osmotic stress. -- Abstract: Osmotic stress is a potent regulator of biological function in many cell types, but its mechanism of action is only partially understood. In this study, we examined whether changes in extracellular osmolality can alter chromatin condensation and the rate of nucleocytoplasmic transport, as potential mechanisms by which osmotic stress can act. Transport of 10 kDa dextran was measured both within and between the nucleus and the cytoplasm using two different photobleaching methods. A mathematical model was developed to describe fluorescence recovery via nucleocytoplasmic transport. As osmolality increased, the diffusion coefficient of dextran decreased in the cytoplasm, but not the nucleus. Hyper-osmotic stress decreased nuclear size and increased nuclear lacunarity, indicating that while the nucleus was getting smaller, the pores and channels interdigitating the chromatin had expanded. The rate of nucleocytoplasmic transport was increased under hyper-osmotic stress but was insensitive to hypo-osmotic stress, consistent with the nonlinear osmotic properties of the nucleus. The mechanism of this osmotic sensitivity appears to be a change in the size and geometry of the nucleus, resulting in a shorter effective diffusion distance for the nucleus. These results may explain physical mechanisms by which osmotic stress can influence intracellular signaling pathways that rely on nucleocytoplasmic transport.

  8. New insights into the mechanisms of mammalian erythroid chromatin condensation and enucleation.

    PubMed

    Ji, Peng

    2015-01-01

    A unique feature in mammalian erythropoiesis is the dramatic chromatin condensation followed by enucleation. This step-by-step process starts at the beginning of terminal erythropoiesis after the hematopoietic stem cells are committed to erythroid lineage. Although this phenomenon is known for decades, the mechanisms of chromatin condensation and enucleation remain elusive. Recent advances in cell and molecular biology have started to reveal the molecular pathways in the regulation of chromatin condensation, the establishment of nuclear polarity prior enucleation, and the rearrangement of actin cytoskeleton in enucleation. However, many challenging questions, especially whether and how the apoptotic mechanisms are involved in chromatin condensation and how to dissect the functions of many actin cytoskeleton proteins in cytokinesis and enucleation, remain to be answered. Here I review our current understanding of mammalian erythroid chromatin condensation and enucleation during terminal differentiation with a focus on more recent studies. I conclude with my perspective of future works in this rising topic in developmental and cell biology.

  9. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure.

    PubMed

    Boitrelle, F; Albert, M; Petit, J-M; Ferfouri, F; Wainer, R; Bergere, M; Bailly, M; Vialard, F; Selva, J

    2013-08-01

    Since an embryo's ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head's area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-19

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

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

    PubMed

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

    2015-05-19

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

  13. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  14. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  15. Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

    PubMed

    Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

    2014-03-01

    Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

  16. Telomere Chromatin Condensation Assay (TCCA): a novel approach to study structural telomere integrity.

    PubMed

    Gonzalez-Vasconcellos, Iria; Alonso-Rodríguez, Silvia; López-Baltar, Isidoro; Fernández, José Luis

    2015-01-01

    Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes are essential for genome stability. Improper higher-order chromatin organization at the chromosome ends can give rise to telomeric recombination and genomic instability. We report the development of an assay to quantify differences in the condensation of telomeric chromatin, thereby offering new opportunities to study telomere biology and stability. We have combined a DNA nuclease digestion with a quantitative PCR (qPCR) assay of telomeric DNA, which we term the Telomere Chromatin Condensation Assay (TCCA). By quantifying the relative quantities of telomeric DNA that are progressively digested with the exonuclease Bal 31 the method can discriminate between different levels of telomeric chromatin condensation. The structural chromatin packaging at telomeres shielded against exonuclease digestion delivered an estimate, which we term Chromatin Protection Factor (CPF) that ranged from 1.7 to 2.3 fold greater than that present in unpacked DNA. The CPF was significantly decreased when cell cultures were incubated with the DNA hypomethylating agent 5-azacytidine, demonstrating the ability of the TCCA assay to discriminate between packaging levels of telomeric DNA.

  17. Proteomics and the genetics of sperm chromatin condensation

    PubMed Central

    Oliva, Rafael; Castillo, Judit

    2011-01-01

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

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

    PubMed

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

    2016-08-23

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

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

    PubMed

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

    2016-08-23

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  2. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

    PubMed

    Golan, R; Shochat, L; Weissenberg, R; Soffer, Y; Marcus, Z; Oschry, Y; Lewin, L M

    1997-01-01

    The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2008-08-21

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

  7. CHROMATIN CONDENSATION IN TERMINALLY DIFFERENTIATING MOUSE ERYTHROBLASTS DOES NOT INVOLVE SPECIAL ARCHITECTURAL PROTEINS BUT DEPENDS ON HISTONE DEACETYLATION

    PubMed Central

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

    2009-01-01

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells. PMID:19172406

  8. Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1.

    PubMed

    Moser, G C; Fallon, R J; Meiss, H K

    1981-02-01

    Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cycle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells; position within the G1 period. Data are presented that deal with three interrelated topics: 1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. 2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1. 3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.

  9. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    PubMed

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics. PMID:24211264

  10. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    PubMed

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27788132

  12. EGFR-mediated chromatin condensation protects KRAS-mutant cancer cells against ionizing radiation.

    PubMed

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

    2014-05-15

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

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

    PubMed Central

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

    2014-01-01

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

  14. Drug-induced premature chromosome condensation (PCC) protocols: cytogenetic approaches in mitotic chromosome and interphase chromatin.

    PubMed

    Gotoh, Eisuke

    2015-01-01

    Chromosome analysis is a fundamental technique which is used in wide areas of cytogenetic study including karyotyping species, hereditary diseases diagnosis, or chromosome biology study. Chromosomes are usually prepared from mitotic cells arrested by colcemid block protocol. However, obtaining mitotic chromosomes is often hampered under several circumstances. As a result, cytogenetic analysis will be sometimes difficult or even impossible in such cases. Premature chromosome condensation (PCC) (see Note 1) is an alternative method that has proved to be a unique and useful way in chromosome analysis. Former, PCC has been achieved following cell fusion method (cell-fusion PCC) mediated either by fusogenic viruses (e.g., Sendai virus) or cell fusion chemicals (e.g., polyethylene glycol), but the cell fusion PCC has several drawbacks. The novel drug-induced PCC using protein phosphatase inhibitors was introduced about 20 years ago. This method is much simpler and easier even than the conventional mitotic chromosome preparation protocol use with colcemid block and furthermore obtained PCC index (equivalent to mitotic index for metaphase chromosome) is usually much higher than colcemid block method. Moreover, this method allows the interphase chromatin to be condensed to visualize like mitotic chromosomes. Therefore drug-induced PCC has opened the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has thus proven the usefulness in cytogenetics and other cell biology fields. For this second edition version, updated modifications/changes are supplemented in Subheadings 2, 3, and 4, and a new section describing the application of PCC in chromosome science fields is added with citation of updated references.

  15. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

    SciTech Connect

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA damage

  16. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

    PubMed Central

    Lee, Y W; Klein, C B; Kargacin, B; Salnikow, K; Kitahara, J; Dowjat, K; Zhitkovich, A; Christie, N T; Costa, M

    1995-01-01

    A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms. PMID:7537850

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  18. Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina.

    PubMed

    Suphamungmee, Worawit; Apisawetakan, Somjai; Weerachatyanukul, Wattana; Wanichanon, Chaitip; Sretarugsa, Prapee; Poomtong, Tanes; Sobhon, Prasert

    2005-02-01

    The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.

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

    PubMed

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

    2006-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Agnieszka, Wojtczak

    2014-10-01

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

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

    PubMed

    Agnieszka, Wojtczak

    2014-10-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2014-12-01

    During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid-liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha. PMID:25262620

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

    SciTech Connect

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

    2012-03-10

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

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

    SciTech Connect

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

    2015-03-13

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-11-01

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

  11. Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle.

    PubMed

    Rahman, Mohammad Bozlur; Kamal, Md Mostofa; Rijsselaere, Tom; Vandaele, Leen; Shamsuddin, Mohammed; Van Soom, Ann

    2014-10-01

    Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n=1239) were collected at three different time points (12, 18 and 24h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P>0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P<0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

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

    SciTech Connect

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

    2004-11-10

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

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

    PubMed

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

    2003-12-01

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

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

    PubMed

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

    2016-07-27

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

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

    PubMed Central

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

    2007-01-01

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

  16. Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1.

    PubMed

    Kliebenstein, Daniel J; Lim, Jackie E; Landry, Laurie G; Last, Robert L

    2002-09-01

    To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.

  17. Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog.

    PubMed

    Garcia-Macias, Vanesa; Martinez-Pastor, Felipe; Alvarez, Mercedes; Garde, Jose Julian; Anel, Enrique; Anel, Luis; de Paz, Paulino

    2006-11-01

    Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.

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

    PubMed

    Rybaczek, Dorota; Maszewski, Janusz

    2007-01-01

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

  19. Disruption of human vigilin impairs chromosome condensation and segregation.

    PubMed

    Wei, Ling; Xie, Xiaoyan; Li, Junhong; Li, Ran; Shen, Wenyan; Duan, Shuwang; Zhao, Rongce; Yang, Wenli; Liu, Qiuying; Fu, Qiang; Qin, Yang

    2015-11-01

    Appropriate packaging and condensation are critical for eukaryotic chromatin's accommodation and separation during cell division. Human vigilin, a multi-KH-domain nucleic acid-binding protein, is associated with alpha satellites of centromeres. DDP1, a vigilin's homolog, is implicated with chromatin condensation and segregation. The expression of vigilin was previously reported to elevate in highly proliferating tissues and increased in a subset of hepatocellular carcinoma patients. Other studies showed that vigilin interacts with CTCF, contributes to regulation of imprinted genes Igf2/H19, and colocalizes with HP1α on heterochromatic satellite 2 and β-satellite repeats. These studies indicate that human vigilin might be involved in chromatin remodeling and regular cell growth. To investigate the potential role of human vigilin in cell cycle, the correlations between vigilin and chromosomal condensation and segregation were studied. Depletion of human vigilin by RNA interference in HepG2 cells resulted in chromosome undercondensation and various chromosomal defects during mitotic phase, including chromosome misalignments, lagging chromosomes, and chromosome bridges. Aberrant polyploid nucleus in telophase was also observed. Unlike the abnormal staining pattern of chromosomes, the shape of spindle was normal. Furthermore, the chromatin showed a greater sensitivity to MNase digestion. Collectively, our findings show that human vigilin apparently participates in chromatin condensation and segregation.

  20. Disruption of human vigilin impairs chromosome condensation and segregation.

    PubMed

    Wei, Ling; Xie, Xiaoyan; Li, Junhong; Li, Ran; Shen, Wenyan; Duan, Shuwang; Zhao, Rongce; Yang, Wenli; Liu, Qiuying; Fu, Qiang; Qin, Yang

    2015-11-01

    Appropriate packaging and condensation are critical for eukaryotic chromatin's accommodation and separation during cell division. Human vigilin, a multi-KH-domain nucleic acid-binding protein, is associated with alpha satellites of centromeres. DDP1, a vigilin's homolog, is implicated with chromatin condensation and segregation. The expression of vigilin was previously reported to elevate in highly proliferating tissues and increased in a subset of hepatocellular carcinoma patients. Other studies showed that vigilin interacts with CTCF, contributes to regulation of imprinted genes Igf2/H19, and colocalizes with HP1α on heterochromatic satellite 2 and β-satellite repeats. These studies indicate that human vigilin might be involved in chromatin remodeling and regular cell growth. To investigate the potential role of human vigilin in cell cycle, the correlations between vigilin and chromosomal condensation and segregation were studied. Depletion of human vigilin by RNA interference in HepG2 cells resulted in chromosome undercondensation and various chromosomal defects during mitotic phase, including chromosome misalignments, lagging chromosomes, and chromosome bridges. Aberrant polyploid nucleus in telophase was also observed. Unlike the abnormal staining pattern of chromosomes, the shape of spindle was normal. Furthermore, the chromatin showed a greater sensitivity to MNase digestion. Collectively, our findings show that human vigilin apparently participates in chromatin condensation and segregation. PMID:26032007

  1. Chromatin Computation

    PubMed Central

    Bryant, Barbara

    2012-01-01

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

  2. Surface nanogratings of abnormal orientation in universal polariton model of laser-induced damage of condensed media

    NASA Astrophysics Data System (ADS)

    Makin, V. S.; Pestov, Yu I.; Makin, R. S.

    2016-08-01

    An experimental results about formation of small-scale gratings of anomalous orientation under the interaction of polarized laser radiation with condensed media were analyzed. The model based on surface polaritons participation in interference process was put forward to explain observed results.

  3. Chromatin Immunoprecipitation.

    PubMed

    Wiehle, Laura; Breiling, Achim

    2016-01-01

    Chromatin immunoprecipitation (ChIP) is a valuable method to investigate protein-DNA interactions in vivo. Since its discovery it has been indispensable to identify binding sites and patterns of a variety of DNA-interacting proteins, such as transcription factors and regulators, modified histones, and epigenetic modifiers. The Polycomb repressors were the first proteins that have been mapped using this technique, which provided the mechanistic basis for the understanding of their biological function. Cross-linked (XChIP) or native (NChIP) chromatin from tissues or cultured cells is fragmented and the protein of interest is immunoprecipitated using a specific antibody. The co-precipitated DNA is then purified and subjected to analysis by region-specific PCR, DNA microarray (ChIP-on-chip), or next-generation sequencing (ChIP-seq). The assay can therefore produce information about the localization of the analyzed protein at specific candidate loci or throughout the entire genome. In this chapter, we provide a detailed protocol of the basic standard ChIP assay and some remarks about variations. PMID:27659971

  4. Interaction of sulfur mustard with rat liver salt fractionated chromatin.

    PubMed

    Jafari, Mahvash; Nateghi, M; Rabbani, A

    2010-01-01

    In this study, the interaction of an alkylating agent, sulfur mustard (SM) with rat liver active (S1 and S2) and inactive (P2) chromatin was investigated employing UV/vis spectroscopy and gel electrophoreses. The results show that SM affects the chromatin structure in a dose-dependent manner. The binding of SM to fractions is different. At lower concentrations (<500 microM), SM seems to unfold the structure and at higher concentrations, it induces aggregation and condensation of chromatin possibly via forming cross-links between the chromatin components. The extent of condensation in S2 is higher when compared to the P2 fraction.

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

    PubMed Central

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

    2016-01-01

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

  6. Mechanical model of the nucleosome and chromatin.

    PubMed

    Bishop, Thomas C; Zhmudsky, Oleksandr O

    2002-04-01

    A theoretical framework for evaluating the approximate energy and dynamic properties associated with the folding of DNA into nucleosomes and chromatin is presented. Experimentally determined elastic constants of linear DNA and a simple fold geometry are assumed in order to derive elastic constants for extended and condensed chromatin. The model predicts the Young s modulus of extended and condensed chromatin to within an order of magnitude of experimentally determined values. Thus we demonstrate that the elastic properties of DNA are a primary determinant of the elastic properties of the higher order folded states. The derived elastic constants are used to predict the speed of propagation of small amplitude waves that excite an extension(sound), twist, bend or shear motion in each folded state. Taken together the results demonstrate that folding creates a hierarchy of time, length and energy scales.

  7. Chromatin Higher-order Structure and Dynamics

    PubMed Central

    Woodcock, Christopher L.; Ghosh, Rajarshi P.

    2010-01-01

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

  8. Unraveling chromatin structure using magnetic tweezers

    NASA Astrophysics Data System (ADS)

    van Noort, John

    2010-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Neutron scattering studies on chromatin higher-order structure

    SciTech Connect

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

    1994-12-31

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

  14. Gearing up chromatin

    PubMed Central

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

    2014-01-01

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

  15. Minor Groove Binder Distamycin Remodels Chromatin but Inhibits Transcription

    PubMed Central

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

    2013-01-01

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

  16. A model for chromatin structure.

    PubMed Central

    Li, H J

    1975-01-01

    A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction. PMID:1101222

  17. Prenucleosomes and Active Chromatin

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Hirakawa, Takeshi; Matsunaga, Sachihiro

    2016-01-01

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

  19. Chromatin signatures of cancer

    PubMed Central

    Morgan, Marc A.; Shilatifard, Ali

    2015-01-01

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

  20. Nucleoporins and chromatin metabolism.

    PubMed

    Ptak, Christopher; Wozniak, Richard W

    2016-06-01

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

  1. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

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

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

    PubMed Central

    Máthé, Csaba; M-Hamvas, Márta; Vasas, Gábor

    2013-01-01

    Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization. PMID:24084787

  3. Chromatin assembly using Drosophila systems.

    PubMed

    Fyodorov, Dmitry V; Levenstein, Mark E

    2002-05-01

    To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP-dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.

  4. Function of sperm chromatin structural elements in fertilization and development

    PubMed Central

    Ward, W. Steven

    2010-01-01

    Understanding how DNA is packaged in the mammalian sperm cell has important implications for human infertility as well as for the cell biology. Recent advances in the study of mammalian sperm chromatin structure and function have altered our perception of this highly condensed, inert chromatin. Sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization. However, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program. The primary levels of the sperm chromatin structure can be divided into three main categories: the large majority of DNA is packaged by protamines, a smaller amount (2–15%) retains histone-bound chromatin and the DNA is attached to the nuclear matrix at roughly 50 kb intervals. Current data suggest that the latter two structural elements are transferred to the paternal pronucleus after fertilization where they have important functional roles. The nuclear matrix organization is essential for DNA replication, and the histone-bound chromatin identifies genes that are important for embryonic development. These data support the emerging view of the sperm genome as providing, in addition to the paternal DNA sequence, a structural framework that includes molecular regulatory factors that are required for proper embryonic development. PMID:19748904

  5. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    PubMed

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage. PMID:11162363

  6. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    PubMed

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

  7. Chromatin fiber allostery and the epigenetic code

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  8. Chromatin and alternative splicing.

    PubMed

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  9. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  10. Polyamines may regulate S-phase progression but not the dynamic changes of chromatin during the cell cycle.

    PubMed

    Laitinen, J; Stenius, K; Eloranta, T O; Hölttä, E

    1998-02-01

    Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. PMID:9443076

  11. Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic apoptotic features.

    PubMed

    Meng, Rui; Xu, Hui-yun; Di, Sheng-meng; Shi, Dong-yan; Qian, Ai-rong; Wang, Jin-fu; Shang, Peng

    2011-02-01

    The aim of the present study was to investigate the effects of abnormal gravity on human mesenchymal stem cells (hMSCs). Strong magnetic field and magnetic field gradient generate a magnetic force that can add to or subtract from the gravitational force. In this study, this is defined as a high-magneto-gravitational environment (HMGE). The HMGE provides three apparent gravity levels, i.e. hypogravity (μg), hypergravity (2g) and normal gravity with strong magnetic field (1g) conditions. After hMSCs were subject to HMGE for 12 h, the proliferation, morphology, structure and apoptosis were investigated. Results showed that the proliferation of hMSCs was inhibited under μg condition. The abnormal gravity induced morphologic characteristics of apoptosis cells, such as cell shrinkage, membrane blebbing, nuclear chromatin condensation and margination, decreased cell viability, and increased caspase-3/7 activity. The rate of apoptosis under μg condition is up to 56.95%. The F-actin stress fibers and microtubules were disrupted under abnormal gravity condition. Under μg-condition, the expression of p53 at mRNA and protein levels was up-regulated more than 9- and 6 folds, respectively. The Pifithrin-α, an specific inhibitor of p53, inhibited the apoptosis and prevented the disruption of cytoskeleton induced by abnormal gravity. These results implied that hMSCs were sensitive to abnormal gravity and exhibited classic apoptotic features, which might be associated with p53 signaling.

  12. Cas9 Functionally Opens Chromatin.

    PubMed

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

    2016-01-01

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

  13. Ochratoxin A at low concentrations inhibits in vitro growth of canine umbilical cord matrix mesenchymal stem cells through oxidative chromatin and DNA damage.

    PubMed

    Rutigliano, Lucia; Valentini, Luisa; Martino, Nicola Antonio; Pizzi, Flavia; Zanghì, Antonina; Dell'Aquila, Maria Elena; Minervini, Fiorenza

    2015-11-01

    Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5×10(-4) to 25nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development.

  14. Presence of histone H2B in Trypanosoma cruzi chromatin.

    PubMed

    Toro, G C; Wernstedt, C; Hellman, U; Galanti, N

    1993-01-01

    The organization of chromatin in protists presents some characteristic features. In Trypanosoma cruzi, no condensation of chromatin into chromosomes is observed during cell division. A systematic characterization of histones should provide information on this peculiar behaviour. Histone H2B from this parasite was characterized by selective dissociation from chromatin in 0.8 M NaCl, by its elution pattern in narrow-bore reversed phase high performance liquid chromatography, by polyacrylamide gel electrophoresis and by partial sequencing of its amino terminal domain. This chromosomal protein differs from histone H2B of other species. The first 12 amino acids are missing which explains its lower molecular weight when compared to human histone H2B. Correspondingly, the amino terminal domain of T. cruzi histone H2B is 25-30% shorter than other histones H2B. Moreover, three out of four acetylation sites present in human histone H2B are missing in T. cruzi histone H2B. The differences in size and in acceptor sites for acetylation of T. cruzi histone H2B when compared to human histone H2B may represent a functional feature to consider for the understanding of the chromatin cycle of condensation in this parasite.

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

    PubMed

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

    2016-01-01

    Distal regulatory elements have been shown to regulate gene transcription through spatial interactions, and single nucleotide polymorphisms (SNPs) are linked with distal gene expression by spatial proximity, which helps to explain the causal role of disease-associated SNPs in non-coding region. Therefore, studies on spatial interactions between chromatin have created a new avenue for elucidating the mechanism of transcriptional regulation in disease pathogenesis. Recently, a growing number of chromatin interactions have been revealed by means of 3C, 4C, 5C, ChIA-PET and Hi-C technologies. To interpret and utilize these interactions, we constructed chromatin-chromatin spatial interaction (CCSI) database by integrating and annotating 91 sets of chromatin interaction data derived from published literature, UCSC database and NCBI GEO database, resulting in a total of 3,017,962 pairwise interactions (false discovery rate < 0.05), covering human, mouse and yeast. A web interface has been designed to provide access to the chromatin interactions. The main features of CCSI are (i) showing chromatin interactions and corresponding genes, enhancers and SNPs within the regions in the search page; (ii) offering complete interaction datasets, enhancer and SNP information in the download page; and (iii) providing analysis pipeline for the annotation of interaction data. In conclusion, CCSI will facilitate exploring transcriptional regulatory mechanism in disease pathogenesis associated with spatial interactions among genes, regulatory regions and SNPs. Database URL: http://songyanglab.sysu.edu.cn/ccsi. PMID:26868054

  16. Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome

    PubMed Central

    Fang, Qianglin; Chen, Ping; Wang, Mingzhu; Fang, Junnan; Yang, Na; Li, Guohong; Xu, Rui-Ming

    2016-01-01

    Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that the specific interaction between IE1-CTD and the H2A-H2B acidic patch impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections. DOI: http://dx.doi.org/10.7554/eLife.11911.001 PMID:26812545

  17. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  18. The use and misuse of sex chromatin screening for 'gender identification' of female athletes.

    PubMed

    de la Chapelle, A

    1986-10-10

    According to the rules of sports organizations such as the International Olympic Committee, competitors registered as females must undergo a "gender verification" test that consists of screening with sex chromatin, followed by further tests in those with an abnormal or inconclusive result. The aims of the gender verification test have not been published but presumably they are to exclude from women's sports events males or other individuals whose muscle strength or body build gives them an unfair advantage over their competitors. It is shown herein that the sex chromatin screening method reveals only a small proportion of such individuals. Moreover, women with certain congenital chromosome abnormalities and other abnormal conditions without increased muscle strength are found to have "abnormal" sex chromatin. Thus, the present screening method is both inaccurate and discriminatory. It is proposed that the aims of gender identification should be defined and methods chosen that achieve the desired result. PMID:3761498

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

    PubMed

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

    2016-03-01

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

  20. Ultrastructure of bovine sperm chromatin.

    PubMed

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

    2015-12-01

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

  1. Single Molecule Studies of Chromatin

    SciTech Connect

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

    2004-01-06

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

  2. Chromatin remodeling in plant development.

    PubMed

    Jarillo, José A; Piñeiro, Manuel; Cubas, Pilar; Martínez-Zapater, José M

    2009-01-01

    Plant development results from specific patterns of gene expression that are tightly regulated in a spatio-temporal manner. Chromatin remodeling plays a central role in establishing these expression patterns and maintaining epigenetic transcriptional states through successive rounds of mitosis that take place within a cell lineage. Plant epigenetic switches occur not only at the embryo stage, but also during postembryonic developmental transitions, suggesting that chromatin remodeling activities in plants can provide a higher degree of regulatory flexibility which probably underlies their developmental plasticity. Here, we highlight recent progress in the understanding of plant chromatin dynamic organization, facilitating the activation or repression of specific sets of genes involved in different developmental programs and integrating them with the response to environmental signals. Chromatin conformation controls gene expression both in actively dividing undifferentiated cells and in those already fate-determined. In this context, we first describe chromatin reorganization activities required to maintain meristem function stable through DNA replication and cell division. Organ initiation at the apex, with emphasis on reproductive development, is next discussed to uncover the chromatin events involved in the establishment and maintenance of expression patterns associated with differentiating cells; this is illustrated with the complex epigenetic regulation of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Finally, we discuss the involvement of chromatin remodeling in plant responses to environmental cues and to different types of stress conditions.

  3. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, A. A. E.; Abdel-Ghaffar, A. E.; Abou El-Roos, M. E. A.; Ahmed, Y. F.

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  4. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa.

    PubMed

    Mahmoud, K Gh M; El-Sokary, A A E; Abdel-Ghaffar, A E; Abou El-Roos, M E A; Ahmed, Y F

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  5. Chromatin Dynamics during Cellular Reprogramming

    PubMed Central

    Apostolou, Effie; Hochedlinger, Konrad

    2014-01-01

    Preface Induced pluripotency is a powerful tool to derive patient-specific stem cells. In addition, it provides a unique assay to study the interplay between transcription factors and chromatin structure. Here, we review the latest insights into chromatin dynamics inherent to induced pluripotency. Moreover, we compare and contrast these events with other physiological and pathological processes involving changes in chromatin and cell state, including germ cell maturation and tumorigenesis. We propose that an integrated view of these seemingly diverse processes could provide mechanistic insights into cell fate transitions in general and might lead to novel approaches in regenerative medicine and cancer treatment. PMID:24153299

  6. Chromatin organization: form to function.

    PubMed

    de Graaf, Carolyn A; van Steensel, Bas

    2013-04-01

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

  7. Chromatin modifications and their function.

    PubMed

    Kouzarides, Tony

    2007-02-23

    The surface of nucleosomes is studded with a multiplicity of modifications. At least eight different classes have been characterized to date and many different sites have been identified for each class. Operationally, modifications function either by disrupting chromatin contacts or by affecting the recruitment of nonhistone proteins to chromatin. Their presence on histones can dictate the higher-order chromatin structure in which DNA is packaged and can orchestrate the ordered recruitment of enzyme complexes to manipulate DNA. In this way, histone modifications have the potential to influence many fundamental biological processes, some of which may be epigenetically inherited. PMID:17320507

  8. CONDENSATION CAN

    DOEpatents

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

    1962-03-01

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

  9. Characterization of an alternative chromatin remodeling to parasperm in a cottid fish, Hemilepidotus gilberti.

    PubMed

    Nakauchi, Yuni; Hayakawa, Youichi; Fujinoki, Masakatsu; Yamamura, Orio; Kobayashi, Makito; Watanabe, Akihiko

    2011-06-01

    The dimorphic sperm of Hemilepidotus gilberti, i.e., haploid eusperm and diploid parasperm, have different morphologies corresponding to their own roles in fertilization. To estimate how these specific sperm morphologies were established, we focused on the nuclear morphologies and examined their changing processes in dimorphic spermiogenesis. Electron microscopic observation revealed that, in euspermatids, chromatin condensation first appeared as a mosaic pattern of moderate electrodense material in the peripheral region of the round nucleus. Those materials spread across the whole area to form a uniformly condensed nucleus. Chromatin condensation began similarly in paraspermatids to that in euspermatids. These became localized to one side of a nucleus and further condensed to form strong electrodense chromatin clusters, which are a specific feature of parasperm. From the remodeled nuclei of eusperm and parasperm, we found five and three kinds of sperm-specific basic proteins (SBPs), respectively, substituted to histones. The N-terminus amino acid sequences of the SBPs suggest that, in parasperm, one major SBP and two minor ones were distinct from each other. In eusperm nuclei, two kinds of specific SBPs were detected in addition to the homologs of parasperm SBPs. The specific SBPs had homologous amino acid sequences with huge arginine clusters, and one of them was most dominant among the five kinds of SBPs. The different combinations of SBPs in the eusperm and parasperm may cause a specific pattern of chromatin condensation in the dimorphic sperm nuclei of H. gilberti.

  10. Craniofacial Abnormalities

    MedlinePlus

    ... of the skull and face. Craniofacial abnormalities are birth defects of the face or head. Some, like cleft ... palate, are among the most common of all birth defects. Others are very rare. Most of them affect ...

  11. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  12. Walking abnormalities

    MedlinePlus

    ... include: Arthritis of the leg or foot joints Conversion disorder (a psychological disorder) Foot problems (such as a ... injuries. For an abnormal gait that occurs with conversion disorder, counseling and support from family members are strongly ...

  13. Nail abnormalities

    MedlinePlus

    Beau's lines; Fingernail abnormalities; Spoon nails; Onycholysis; Leukonychia; Koilonychia; Brittle nails ... Just like the skin, the fingernails tell a lot about your health: ... the fingernail. These lines can occur after illness, injury to ...

  14. Vernalization-mediated chromatin changes.

    PubMed

    Zografos, Brett R; Sung, Sibum

    2012-07-01

    Proper flowering time is vital for reproductive fitness in flowering plants. In Arabidopsis, vernalization is mediated primarily through the repression of a MADS box transcription factor, FLOWERING LOCUS C (FLC). The induction of a plant homeodomain-containing protein, VERNALIZATION INSENSITIVE 3 (VIN3), by vernalizing cold is required for proper repression of FLC. One of a myriad of changes that occurs after VIN3 is induced is the establishment of FLC chromatin at a mitotically repressed state due to the enrichment of repressive histone modifications. VIN3 induction by cold is the earliest known event during the vernalization response and includes changes in histone modifications at its chromatin. Here, the current understanding of the vernalization-mediated chromatin changes in Arabidopsis is discussed, with a focus on the roles of shared chromatin-modifying machineries in regulating VIN3 and FLC gene family expression during the course of vernalization.

  15. Painting a Clearer Picture of Chromatin.

    PubMed

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

    2016-02-22

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

  16. Nuclear abnormalities in buccal mucosa cells of patients with type I and II diabetes treated with folic acid.

    PubMed

    Gómez-Meda, B C; Zamora-Perez, A L; Muñoz-Magallanes, T; Sánchez-Parada, M G; García Bañuelos, J J; Guerrero-Velázquez, C; Sánchez-Orozco, L V; Vera-Cruz, J M; Armendáriz-Borunda, J; Zúñiga-González, G M

    2016-02-01

    Diabetes mellitus (DM) is characterized by high blood glucose. Excessive production of free radicals may cause oxidative damage to DNA and other molecules, leading to complications of the disease. It may be possible to delay or reduce such damage by administration of antioxidants such as folic acid (FA). The objective of this study was to determine the effect of FA on nuclear abnormalities (NAs) in the oral mucosa of patients with DM. NAs (micronucleated cells, binucleated cells, pyknotic nuclei, karyorrhexis, karyolysis, abnormally condensed chromatin, and nuclear buds) were analyzed in 2000 cells from 45 healthy individuals (control group) and 55 patients with controlled or uncontrolled type I or II DM; 35 patients in the latter group were treated with FA. Samples were taken from the FA group before and after treatment. An increased rate of NAs was found in patients with DM in comparison with that of the control group (P<0.001). FA supplementation in patients with DM reduced the frequency of NAs (20.4 ± 8.0 before treatment vs. 10.5 ± 5.2 after treatment; P<0.001). The type I and type II DM and controlled and uncontrolled DM subgroups were analyzed in terms of sex, age, and smoking habit. The significantly reduced frequencies of buccal mucosa cells with micronuclei, binucleation, pyknosis, karyorrhexis, karyorrhexis+abnormally condensed chromatin, karyolysis, and nuclear buds produced by FA supplementation in DM patients (P<0.02) are consistent with the idea that free radicals are responsible for the increased frequency of NAs in DM patients.

  17. Nuclear abnormalities in buccal mucosa cells of patients with type I and II diabetes treated with folic acid.

    PubMed

    Gómez-Meda, B C; Zamora-Perez, A L; Muñoz-Magallanes, T; Sánchez-Parada, M G; García Bañuelos, J J; Guerrero-Velázquez, C; Sánchez-Orozco, L V; Vera-Cruz, J M; Armendáriz-Borunda, J; Zúñiga-González, G M

    2016-02-01

    Diabetes mellitus (DM) is characterized by high blood glucose. Excessive production of free radicals may cause oxidative damage to DNA and other molecules, leading to complications of the disease. It may be possible to delay or reduce such damage by administration of antioxidants such as folic acid (FA). The objective of this study was to determine the effect of FA on nuclear abnormalities (NAs) in the oral mucosa of patients with DM. NAs (micronucleated cells, binucleated cells, pyknotic nuclei, karyorrhexis, karyolysis, abnormally condensed chromatin, and nuclear buds) were analyzed in 2000 cells from 45 healthy individuals (control group) and 55 patients with controlled or uncontrolled type I or II DM; 35 patients in the latter group were treated with FA. Samples were taken from the FA group before and after treatment. An increased rate of NAs was found in patients with DM in comparison with that of the control group (P<0.001). FA supplementation in patients with DM reduced the frequency of NAs (20.4 ± 8.0 before treatment vs. 10.5 ± 5.2 after treatment; P<0.001). The type I and type II DM and controlled and uncontrolled DM subgroups were analyzed in terms of sex, age, and smoking habit. The significantly reduced frequencies of buccal mucosa cells with micronuclei, binucleation, pyknosis, karyorrhexis, karyorrhexis+abnormally condensed chromatin, karyolysis, and nuclear buds produced by FA supplementation in DM patients (P<0.02) are consistent with the idea that free radicals are responsible for the increased frequency of NAs in DM patients. PMID:26921015

  18. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  19. A model for the structure of chromatin in mammalian sperm

    PubMed Central

    1982-01-01

    DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences. PMID:7096440

  20. Chromatin beacons: global sampling of chromatin physical properties using chromatin charting lines.

    PubMed

    Amini, Aniça; Luo, Chongyuan; Lam, Eric

    2011-01-01

    The extent to which physical properties and intranuclear locations of chromatin can influence transcription output remains unclear and poorly quantified. Because the scale and resolution at which structural parameters can be queried are usually so different from the scale that transcription outputs are measured, the integration of these data is often indirect. To overcome this limitation in quantifying chromatin structural parameters at different locations in the genome, a Chromatin Charting collection with 277 transposon-tagged Arabidopsis lines has been established in order to discover correlations between gene expression and the physical properties of chromatin loci within the nuclei. In these lines, dispersed loci in the Arabidopsis genome are tagged with an identical transgene cassette containing a luciferase gene reporter, which permits the quantification of gene expressions in real time, and an ∼2 kb LacO repeat that acts as a "chromatin beacon" to facilitate the visual tracking of a tagged locus in living plants via the expression of LacI-GFP fusion proteins in trans. In this chapter, we describe the methods for visualizing and tracking these insertion loci in vivo and illustrate the potential of using this approach to correlate chromatin mobility with gene expression in living plants.

  1. Chromatin pattern by variogram analysis.

    PubMed

    Diaz, G; Zucca, A; Setzu, M D; Cappai, C

    1997-11-01

    Many cytological processes such as cell proliferation, differentiation, transformation, apoptosis, etc., are accompanied by specific chromatin changes, usually identified on the basis of the relative content of euchromatin and heterochromatin. In order to achieve a quantitative, non-subjective evaluation of the chromatin pattern, two different approaches may be undertaken, one consisting in the analysis of the several morphological features of chromatin grains (size, shape, density, arrangement, and distribution), and the second consisting in the analysis of the chromatin globally considered as a coherent texture. Although the second approach appears to be simpler and more suitable, methods of texture analysis--including those specifically designed for the analysis of the chromatin pattern--are rarely applied due mainly to the unsuitability of sampling procedures and the excessive crypticism of results. As an alternative to traditional texture analysis, we suggest a method supported by a sound mathematical theory and approximately 30 years of applications in the field of geostatistics. The method, called variogram, analyzes the intrinsic structure of data sampled at different distance intervals and directions, and outputs easily understandable results. Recently, variogram analysis has successfully been exported from geostatistics to other fields (for example, ecology and epidemiology) that make use of spatially referenced variables. Based on the fact that pixels represent a perfect array of data ordered at regular distance intervals and directions, the variogram can be adopted to explore nuclear images and recognize chromatin patterns. Variograms of different nuclei can be summarized by multivariate methods without the need of previous standardization of data. This allows comparison and discrimination of chromatin patterns from mixed cell populations. Preliminary data obtained from young neurons undergoing massive apoptosis reveal a self-consistent map of nuclear

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

    PubMed

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

    2014-01-01

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

  3. The integrity of sperm chromatin in young tropical composite bulls.

    PubMed

    Fortes, M R S; Holroyd, R G; Reverter, A; Venus, B K; Satake, N; Boe-Hansen, G B

    2012-07-15

    Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r=0.34, P=0.0076), Mot (r=0.36, P=0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r=0.31, P=0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r=-0.28, P=0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r=-0.53, P<0.0001), the percentage of sperm with head abnormalities (r=0.68, P<0.0001) and the percentage of intact sperm (Int) with SBH (r=-0.26, P=0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age. PMID:22494672

  4. Single Molecule Studies of Chromatin

    SciTech Connect

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

    2006-02-06

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

  5. Chromatin Preparation and Chromatin Immuno-precipitation from Drosophila Embryos.

    PubMed

    Löser, Eva; Latreille, Daniel; Iovino, Nicola

    2016-01-01

    This protocol provides specific details on how to perform Chromatin immunoprecipitation (ChIP) from Drosophila embryos. ChIP allows the matching of proteins or histone modifications to specific genomic regions. Formaldehyde-cross-linked chromatin is isolated and antibodies against the target of interest are used to determine whether the target is associated with a specific DNA sequence. This can be performed in spatial and temporal manner and it can provide information about the genome-wide localization of a given protein or histone modification if coupled with deep sequencing technology (ChIP-Seq). PMID:27659972

  6. Chromatin remodeling in nuclear cloning.

    PubMed

    Wade, Paul A; Kikyo, Nobuaki

    2002-05-01

    Nuclear cloning is a procedure to create new animals by injecting somatic nuclei into unfertilized oocytes. Recent successes in mammalian cloning with differentiated adult nuclei strongly indicate that oocyte cytoplasm contains unidentified remarkable reprogramming activities with the capacity to erase the previous memory of cell differentiation. At the heart of this nuclear reprogramming lies chromatin remodeling as chromatin structure and function define cell differentiation through regulation of the transcriptional activities of the cells. Studies involving the modification of chromatin elements such as selective uptake or release of binding proteins, covalent histone modifications including acetylation and methylation, and DNA methylation should provide significant insight into the molecular mechanisms of nuclear dedifferentiation and redifferentiation in oocyte cytoplasm.

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  9. Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.

    PubMed

    Gogol, P; Bochenek, M; Smorag, Z

    2000-12-01

    The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production

  10. APPLICATION OF THE SPERM CHROMATIN STRUCTURE ASSAY TO THE TEPLICE PROGRAM SEMEN STUDIES: A NEW METHOD FOR EVALUATING SPERM NUCLEAR CHROMATIN DAMAGE

    EPA Science Inventory

    ABSTRACT
    A 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...

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

    PubMed

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

    2014-12-01

    It remains unclear how the 2m of human genomic DNA is organized in each cell. The textbook model has long assumed that the 11-nm-diameter nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, is folded into a 30-nm chromatin fiber. One of the classical models assumes that the 30-nm chromatin fiber is further folded helically to form a larger fiber. Small-angle X-ray scattering (SAXS) is a powerful method for investigating the bulk structure of interphase chromatin and mitotic chromosomes. SAXS can detect periodic structures in biological materials in solution. In our SAXS results, no structural feature larger than 11 nm was detected. Combining this with a computational analysis of "in silico condensed chromatin" made it possible to understand more about the X-ray scattering profiles and suggested that the chromatin in interphase nuclei and mitotic chromosomes essentially consists of irregularly folded nucleosome fibers lacking the 30-nm chromatin structure. In this article, we describe the experimental details of our SAXS and modeling systems. We also discuss other methods for investigating the chromatin structure in cells. PMID:25168089

  12. Structural and functional genome analysis using extended chromatin

    SciTech Connect

    Heaf, T.; Ward, D.C.

    1994-09-01

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

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

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

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

  14. Condensation polyimides

    NASA Technical Reports Server (NTRS)

    Hergenrother, P. M.

    1989-01-01

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

  15. Condensation of nonstochiometric DNA/polycation complexes by divalent cations.

    PubMed

    Budker, Vladimir; Trubetskoy, Vladimir; Wolff, Jon A

    2006-12-15

    This study found that divalent cations induced the further condensation of partially condensed DNA within nonstochiometric polycation complexes. The addition of a few mmol of a divalent cation such as calcium reduced by half the inflection point at which DNA became fully condensed by poly-L-lysine (PLL) and a variety of other polycations. The effect on DNA condensation was initially observed using a new method, which is based on the concentration-dependent self-quenching of fluorescent moieties (e.g., rhodamine) covalently linked to the DNA backbone at relatively high densities. Additional analyses, which employed ultracentrifugation, dynamic light scattering, agarose gel electrophoresis, and atomic force microscopy, confirmed the effect of divalent cations. These results provide an additional accounting of the process by which divalent cations induce greater chromatin compaction that is based on the representation of chromatin fibers as a nonstoichiometric polyelectrolyte complex. They also offer a new approach to assemble nonviral vectors for gene therapy.

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

    PubMed Central

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

    1974-01-01

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

  17. A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis.

    PubMed

    Schellhaus, Anna K; Magalska, Adriana; Schooley, Allana; Antonin, Wolfram

    2015-12-19

    During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

  18. Organisation of subunits in chromatin.

    PubMed

    Carpenter, B G; Baldwin, J P; Bradbury, E M; Ibel, K

    1976-07-01

    There is considerable current interest in the organisation of nucleosomes in chromatin. A strong X-ray and neutron semi-meridional diffraction peak at approximately 10 nm had previously been attributed to the interparticle specing of a linear array of nucleosomes. This diffraction peak could also result from a close packed helical array of nucleosomes. A direct test of these proposals is whether the 10 nm peak is truly meridional as would be expected for a linear array of nucleosomes or is slightly off the meridian as expected for a helical array. Neutron diffraction studies of H1-depleted chromatin support the latter alternative. The 10 nm peak has maxima which form a cross-pattern with semi-meridional angle of 8 to 9 degrees. This is consistent with a coil of nucleosomes of pitch 10 nm and outer diameter of approximately 30 nm. These dimensions correspond to about six nucleosomes per turn of the coli.

  19. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1998-11-01

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

  1. Changes in large-scale chromatin structure and function during oogenesis: a journey in company with follicular cells.

    PubMed

    Luciano, Alberto M; Franciosi, Federica; Dieci, Cecilia; Lodde, Valentina

    2014-09-01

    The mammalian oocyte nucleus or germinal vesicle (GV) exhibits characteristic chromatin configurations, which are subject to dynamic modifications through oogenesis. Aim of this review is to highlight how changes in chromatin configurations are related to both functional and structural modifications occurring in the oocyte nuclear and cytoplasmic compartments. During the long phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. The decondensed configuration of chromatin then undergoes profound rearrangements during the final stages of oocyte growth that are tightly associated with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional interaction with the follicular cells, a connection between cumulus cells gene expression profile and oocyte developmental competence, according to chromatin configuration is proposed. This analysis can help in identifying candidate genes involved in the process of oocyte developmental competence acquisition and in providing non-invasive biomarkers of oocyte health status that can have important implications in treating human infertility as well as managing breeding schemes in domestic mammals.

  2. Polariton condensates

    SciTech Connect

    Snoke, David; Littlewood, Peter

    2010-08-15

    Most students of physics know about the special properties of Bose-Einstein condensates (BECs) as demonstrated in the two best-known examples: superfluid helium-4, first reported in 1938, and condensates of trapped atomic gases, first observed in 1995. (See the article by Wolfgang Ketterle in PHYSICS TODAY, December 1999, page 30.) Many also know that superfluid {sup 3}He and superconducting metals contain BECs of fermion pairs. An underlying principle of all those condensed-matter systems, known as quantum fluids, is that an even number of fermions with half-integer spin can be combined to make a composite boson with integer spin. Such composite bosons, like all bosons, have the property that below some critical temperature--roughly the temperature at which the thermal de Broglie wavelength becomes comparable to the distance between the bosons--the total free energy is minimized by having a macroscopic number of bosons enter a single quantum state and form a macroscopic, coherent matter wave. Remarkably, the effect of interparticle repulsion is to lead to quantum mechanical exchange interactions that make that state robust, since the exchange interactions add coherently.

  3. Imaging of DNA/Nanosphere Condensates

    NASA Astrophysics Data System (ADS)

    Krishnan, R.

    2005-03-01

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

  4. Assessment of nuclear abnormalities in exfoliated cells from the oral epithelium of mobile phone users.

    PubMed

    Souza, Leonardo da Cunha Menezes; Cerqueira, Eneida de Moraes Marcílio; Meireles, José Roberto Cardoso

    2014-06-01

    Transmission and reception of mobile telephony signals take place through electromagnetic wave radiation, or electromagnetic radiofrequency fields, between the mobile terminal and the radio base station. Based on reports in the literature on adverse effects from exposure to this type of radiation, the objective of this study was to evaluate the genotoxic and cytotoxic potential of such exposure, by means of the micronucleus test on exfoliated cells from the oral epithelium. The sample included 45 individuals distributed in 3 groups according to the amount of time in hours per week (t) spent using mobile phones: group I, t > 5 h; group II, t > 1 h and ≤ 5 h; and group III, t ≤ 1 h. Cells from the oral mucosa were analyzed to assess the numbers of micronuclei, broken egg structures and degenerative nuclear abnormalities indicative of apoptosis (condensed chromatin, karyorrhexis and pyknosis) or necrosis (karyolysis in addition to these changes). The occurrences of micronuclei and degenerative nuclear abnormalities did not differ between the groups, but the number of broken egg (structures that may be associated with gene amplification) was significantly greater in the individuals in group I (p < 0.05).

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. Diet-mediated alteration of chromatin structure.

    PubMed

    Castro, C E; Armstrong-Major, J; Ramirez, M E

    1986-08-01

    Higher-order chromatin structure and the process of transcription are related. The significance of a nutritional state's altering chromatin structure lies in the potential role of that nutritional state in the regulation of gene expression. In rats short-term feeding of semisynthetic diets varying in the proportion of carbohydrate, protein, or fat alters the configuration of liver chromatin as measured by sensitivity to micrococcal nuclease (EC 3.1.31.1). A carbohydrate-rich, fat-free diet increases the sensitivity of rat liver chromatin to micrococcal nuclease and decreases the nucleosome repeat length. In contrast, a protein-free diet or a diet deficient in magnesium or zinc decreases the sensitivity of liver nuclear chromatin to micrococcal nuclease. Diet-mediated mechanisms that alter chromatin structure are now unknown, but the continued study of nutritional interaction with the genome should identify the responsible features as well as their significance to gene function.

  8. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2012-01-01

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

  10. Chromatin Remodelers: From Function to Dysfunction.

    PubMed

    Längst, Gernot; Manelyte, Laura

    2015-01-01

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

  11. Open chromatin reveals the functional maize genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Expression-Dependent Folding of Interphase Chromatin

    PubMed Central

    Jerabek, Hansjoerg; Heermann, Dieter W.

    2012-01-01

    Multiple studies suggest that chromatin looping might play a crucial role in organizing eukaryotic genomes. To investigate the interplay between the conformation of interphase chromatin and its transcriptional activity, we include information from gene expression profiles into a polymer model for chromatin that incorporates genomic loops. By relating loop formation to transcriptional activity, we are able to generate chromosome conformations whose structural and topological properties are consistent with experimental data. The model particularly allows to reproduce the conformational variations that are known to occur between highly and lowly expressed chromatin regions. As previously observed in experiments, lowly expressed regions of the simulated polymers are much more compact. Due to the changes in loop formation, the distributions of chromatin loops are also expression-dependent and exhibit a steeper decay in highly active regions. As a results of entropic interaction between differently looped parts of the chromosome, we observe topological alterations leading to a preferential positioning of highly transcribed loci closer to the surface of the chromosome territory. Considering the diffusional behavior of the chromatin fibre, the simulations furthermore show that the higher the expression level of specific parts of the chromatin fibre is, the more dynamic they are. The results exhibit that variations of loop formation along the chromatin fibre, and the entropic changes that come along with it, do not only influence the structural parameters on the local scale, but also effect the global chromosome conformation and topology. PMID:22649534

  13. A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies.

    PubMed

    Schaner, Christine E; Deshpande, Girish; Schedl, Paul D; Kelly, William G

    2003-11-01

    In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.

  14. Control of RNA synthesis by chromatin proteins.

    PubMed Central

    Cedar, H; Solage, A; Zurucki, F

    1976-01-01

    The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA. PMID:787926

  15. Condensation heat transfer

    NASA Astrophysics Data System (ADS)

    Rose, J. W.

    The paper gives a brief description of some of the better understood aspects of condensation heat transfer and includes discussion of the liquid-vapour interface, natural and forced convection laminar film condensation and dropwise condensation.

  16. Inheritance of epigenetic chromatin silencing

    PubMed Central

    David-Rus, Diana; Mukhopadhyay, Swagatam; Lebowitz, Joel L.; Sengupta, Anirvan M.

    2010-01-01

    Maintenance of alternative chromatin states through cell divisions pose some fundamental constraints on the dynamics of histone modifications. In this paper, we study the systems biology of epigenetic inheritance by defining and analyzing general classes of mathematical models. We discuss how the number of modification states involved plays an essential role in the stability of epigenetic states. In addition, DNA duplication and the consequent dilution of marked histones act as a large perturbation for a stable state of histone modifications. The requirement that this large perturbation falls into the basin of attraction of the original state sometimes leads to additional constraints on effective models. Two such models, inspired by two different biological systems, are compared in their fulfilling the requirements of multistability and of recovery after DNA duplication. We conclude that in the presence of multiple histone modifications that characterize alternative epigenetic stable states, these requirements are more easily fulfilled. PMID:19174167

  17. Chromatin modifications associated with diabetes.

    PubMed

    Keating, Samuel T; El-Osta, Assam

    2012-08-01

    Accelerated rates of vascular complications are associated with diabetes mellitus. Environmental factors including hyperglycaemia contribute to the progression of diabetic complications. Epidemiological and experimental animal studies identified poor glycaemic control as a major contributor to the development of complications. These studies suggest that early exposure to hyperglycaemia can instigate the development of complications that present later in the progression of the disease, despite improved glycaemic control. Recent experiments reveal a striking commonality associated with gene-activating hyperglycaemic events and chromatin modification. The best characterised to date are associated with the chemical changes of amino-terminal tails of histone H3. Enzymes that write specified histone tail modifications are not well understood in models of hyperglycaemia and metabolic memory as well as human diabetes. The best-characterised enzyme is the lysine specific Set7 methyltransferase. The contribution of Set7 to the aetiology of diabetic complications may extend to other transcriptional events through methylation of non-histone substrates. PMID:22639343

  18. Computational strategies to address chromatin structure problems

    NASA Astrophysics Data System (ADS)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

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

  19. Sodium butyrate induced structural changes in HeLa cell chromatin

    SciTech Connect

    Reczek, P.R.; Weissman, D.; Huvos, P.E.; Fasman, G.D.

    1982-01-01

    Postsynthetic modifications of core histones by treatment of HeLa S3 cells with 5 mM sodium butyrate lead to alterations in the structure of high molecular weight chromatin. Whole chromatin from butyrate-treated cells, which results in highly acetylated core histones, has an ellipticity (theta)/sub 282.5/ of 3700 deg cm/sup 2/ dmol/sup -1/ (0.2 mM EDTA, pH 7.4) that is 1200 deg cm/sup 2/ dmol/sup -1/ less than chromatin from untreated HeLa cells, suggesting a more condensed structure. No difference in the circular dichroism spectra was observed in Hl-stripped, high molecular weight chromatin obtained from control and butyrate-treated cells at low (0.2 mM EDTA, pH 7.4) ionic strength.Thermal denaturation profiles of high molecular weight chromatin were resolved into three transitions and exhibited a shifting of hyperchromicity from transition I to transition III, at a higher T/sub m/, with butyrate treatment of HeLa cells, further indicating a more compact structure. Thermal denaturation profiles of Hl-stripped chromatin were not affected by butyrate treatment. Ionic strength studies in the range of 0-5 mM NaH/sub 2/PO/sub 4/, 0.2 mM EDTA, pH 7.4, of high molecular weight chromatin exhibited a decrease in (theta)/sub 282.5/ and a shifting of hyperchromicity from transition I to transition III with increasing ionic strength. Control high molecular weight chromatin was more sensitive to changes in ionic strength than its highly acetylated counterpart. These results suggest that acetylation of histones alone does not result in a change in histone-DNA interaction but other changes associated with butyrate treatment most probably cause a more condensed structure, of the fraction studied herein, which is mediated by Hl or other materials removed during stripping in 0.35 M NaCl.

  20. Sodium butyrate induced structural changes in HeLa cell chromatin

    SciTech Connect

    Reczek, P.R.; Weissman, D.; Huvos, P.E.; Fasman, G.D.

    1982-03-02

    Postsynthetic modifications of core histones by treatment of HeLa S3 cells with 5 mM sodium butyrate lead to alterations in the structure of high molecular weight chromatin. Whole chromatin from butyrate-treated cells, which results in highly acetylated core histones, has an ellipticity (THETA)/sub 282.5/ of 3700 deg cm/sup 2/ dmol/sup -1/ (0.2 mM EDTA, pH 7.4) that is 1200 deg cm/sup 2/ dmol/sup -1/ less than chromatin from untreated HeLa cells, suggesting a more condensed structure. No difference in the circular dichroism spectra was observed in Hl-stripped, high molecular weight chromatin obtained from control and butyrate-treated cells at low (0.2 mM EDTA, pH 7.4) ionic strength. Thermal denaturation profiles of high molecular weight chromatin were resolved into three transitions and exhibited a shifting of hyperchromicity from transition I to transition III, at a higher T/sub m/, with butyrate treatment of HeLa cells, further indicating a more compact structure. Thermal denaturation profiles of Hl-stripped chromatin were not affected by butyrate treatment. Ionic strength studies in the range of 0-5 mM NaH/sub 2/PO/sub 4/, 0.2 mM EDTA, pH 7.4, of high molecular weight chromatin exhibited a decrease in (THETA)/sub 282.5/ and shifting of hyperchromicity from transition I to transition III with increasing ionic strength. Control high molecular weight chromatin was more sensitive to changes in ionic strength than its highly acetylated counterpart. These results suggest that acetylation of histones alone does not result in a change in histone-DNA interaction but other changes associated with butyrate treatment most probably cause a more condensed structure, of the fraction studied herein, which is mediated by Hl or other materials removed during stripping in 0.35 M NaCl.

  1. Chromatin maintenance by a molecular motor protein

    PubMed Central

    Sung, Myong-Hee; Misteli, Tom

    2011-01-01

    The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure. PMID:22130187

  2. Targeting of cohesin by transcriptionally silent chromatin.

    PubMed

    Chang, Chuang-Rung; Wu, Ching-Shyi; Hom, Yolanda; Gartenberg, Marc R

    2005-12-15

    Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids. PMID:16319193

  3. Chromatin remodelling initiation during human spermiogenesis

    PubMed Central

    De Vries, Marieke; Ramos, Liliana; Housein, Zjwan; De Boer, Peter

    2012-01-01

    Summary During the last phase of spermatogenesis, spermiogenesis, haploid round spermatids metamorphose towards spermatozoa. Extensive cytoplasmic reduction and chromatin remodelling together allow a dramatic decrease of cellular, notably nuclear volume. DNA packing by a nucleosome based chromatin structure is largely replaced by a protamine based one. At the cytoplasmic level among others the acrosome and perinuclear theca (PNT) are formed. In this study we describe the onset of chromatin remodelling to occur concomitantly with acrosome and PNT development. In spread human round spermatid nuclei, we show development of a DAPI-intense doughnut-like structure co-localizing with the acrosomal sac and sub acrosomal PNT. At this structure we observe the first gradual decrease of nucleosomes and several histones. Histone post-translational modifications linked to chromatin remodelling such as H4K8ac and H4K16ac also delineate the doughnut, that is furthermore marked by H3K9me2. During the capping phase of acrosome development, the size of the doughnut-like chromatin domain increases, and this area often is marked by uniform nucleosome loss and the first appearance of transition protein 2 and protamine 1. In the acrosome phase at nuclear elongation, chromatin remodelling follows the downward movement of the marginal ring of the acrosome. Our results indicate that acrosome development and chromatin remodelling are interacting processes. In the discussion we relate chromatin remodelling to the available data on the nuclear envelope and the linker of nucleoskeleton and cytoskeleton (LINC) complex of spermatids, suggesting a signalling route for triggering chromatin remodelling. PMID:23213436

  4. Condensation model for the ESBWR passive condensers

    SciTech Connect

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

    2012-07-01

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

  5. Chromatin and the genome integrity network

    PubMed Central

    Papamichos-Chronakis, Manolis; Peterson, Craig L.

    2013-01-01

    The maintenance of genome integrity is essential for organism survival and for the inheritance of traits to offspring. Genomic instability is caused by DNA damage, aberrant DNA replication or uncoordinated cell division, which can lead to chromosomal aberrations and gene mutations. Recently, chromatin regulators that shape the epigenetic landscape have emerged as potential gatekeepers and signalling coordinators for the maintenance of genome integrity. Here, we review chromatin functions during the two major pathways that control genome integrity: namely, repair of DNA damage and DNA replication. We also discuss recent evidence that suggests a novel role for chromatin-remodelling factors in chromosome segregation and in the prevention of aneuploidy. PMID:23247436

  6. Chromatin Fiber Dynamics under Tension and Torsion

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

    PubMed

    Citterio, E; Van Den Boom, V; Schnitzler, G; Kanaar, R; Bonte, E; Kingston, R E; Hoeijmakers, J H; Vermeulen, W

    2000-10-01

    The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed. PMID:11003660

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

    SciTech Connect

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

    2005-01-15

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

  10. Modeling studies of chromatin fiber structure as a function of DNA linker length

    PubMed Central

    Perišić, Ognjen; Collepardo-Guevara, Rosana; Schlick, Tamar

    2010-01-01

    Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRL) of the linker DNA connecting the nucleosomes. While single cellular organisms and rapidly growing cells with high protein production have short NRL ranging from 160 to 189 base pairs (bp), mature cells usually have longer NRL ranging between 190 and 220 bp. Recently, various experimental studies have examined the effect of NRL on the internal organization of chromatin fiber. Here we investigate by mesoscale modeling of oligonucleosomes the folding patterns for different NRL, with and without linker histone, under typical monovalent salt conditions using both one-start solenoid and two-start zigzag starting configurations. We find that short to medium NRL chromatin fibers (173 to 209 bp) with linker histone condense into irregular zigzag structures, and that solenoid-like features are viable only for longer NRL (226 bp). We suggest that medium NRL are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRL) fold into narrow fibers, while the latter (long NRL) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the linker histone has a small effect on the condensation of short-NRL arrays but an important condensation effect on medium-NRL arrays which have linker lengths similar to the linker histone lengths. Finally, we suggest that the medium-NRL species, with densely packed fiber arrangements, may be advantageous for epigenetic control because their histone tail modifications can have a greater effect compared to other fibers due to their more extensive nucleosome interaction network. PMID:20709077

  11. Expanding the roles of chromatin insulators in nuclear architecture, chromatin organization and genome function.

    PubMed

    Schoborg, Todd; Labrador, Mariano

    2014-11-01

    Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior.

  12. Chromatin Domains: The Unit of Chromosome Organization.

    PubMed

    Dixon, Jesse R; Gorkin, David U; Ren, Bing

    2016-06-01

    How eukaryotic chromosomes fold inside the nucleus is an age-old question that remains unanswered today. Early biochemical and microscopic studies revealed the existence of chromatin domains and loops as a pervasive feature of interphase chromosomes, but the biological implications of such organizational features were obscure. Genome-wide analysis of pair-wise chromatin interactions using chromatin conformation capture (3C)-based techniques has shed new light on the organization of chromosomes in interphase nuclei. Particularly, the finding of cell-type invariant, evolutionarily conserved topologically associating domains (TADs) in a broad spectrum of cell types has provided a new molecular framework for the study of animal development and human diseases. Here, we review recent progress in characterization of such chromatin domains and delineation of mechanisms of their formation in animal cells. PMID:27259200

  13. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

    PubMed

    Zhang, Yang; Máté, Gabriell; Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W

    2015-01-01

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

  19. Chromatin remodelers: We are the drivers!!

    PubMed

    Tyagi, Monica; Imam, Nasir; Verma, Kirtika; Patel, Ashok K

    2016-07-01

    Chromatin is a highly dynamic structure that imparts structural organization to the genome and regulates the gene expression underneath. The decade long research in deciphering the significance of epigenetics in maintaining cellular integrity has embarked the focus on chromatin remodeling enzymes. These drivers have been categorized as readers, writers and erasers with each having significance of their own. Largely, on the basis of structure, ATP dependent chromatin remodelers have been grouped into 4 families; SWI/SNF, ISWI, IN080 and CHD. It is still unclear to what degree these enzymes are swayed by local DNA sequences when shifting a nucleosome to different positions. The ability of regulating active and repressive transcriptional state via open and close chromatin architecture has been well studied however, the significance of chromatin remodelers in regulating transcription at each step i.e. initiation, elongation and termination require further attention. The authors have highlighted the significance and role of different chromatin remodelers in transcription, DNA repair and histone variant deposition. PMID:27429206

  20. Links between genome replication and chromatin landscapes.

    PubMed

    Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2015-07-01

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

  1. Chromatin structure in scrapie and Alzheimer's disease.

    PubMed

    McLachlan, D R; Lukiw, W J; Cho, H J; Carp, R I; Wisniewski, H

    1986-11-01

    Scrapie affected brains exhibit a number of pathological features in common with the human neurodegenerative condition, Alzheimer's disease. The present report describes studies on chromatin structure seen in these two disease processes. Chromatin associated proteins influence transcriptional activity of DNA through an effect upon chromatin structure. We examined chromatin structure by: measuring the capacity of the enzyme micrococcal nuclease to release mono- and dinucleosomes from isolated nuclei and measuring DNA-histone interactions by examining the effect of ambient tonicity upon the release of chromatin proteins. In two strains of mice infected with two strains of scrapie agent there was reduced accessibility to micrococcal nuclease and an increased content on dinucleosomes of the histone H1 and H1(0) types. These changes precede clinical signs of scrapie and resemble those found in the human conditions of Alzheimer's and Pick's disease. Scrapie mouse brain differs from Alzheimer brain in that scrapie does not alter histone-DNA interactions as monitored by ionically induced histone release from chromatin. Despite similarities, the scrapie agent appears to operate upon different molecular mechanisms than those found in Alzheimer's disease.

  2. Chromatin insulation by a transcriptional activator

    PubMed Central

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

    2003-01-01

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

  3. Epigenomic regulation of oncogenesis by chromatin remodeling.

    PubMed

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

    2016-08-25

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

  4. Mutations in chromatin machinery and pediatric high-grade glioma.

    PubMed

    Lulla, Rishi R; Saratsis, Amanda Muhs; Hashizume, Rintaro

    2016-03-01

    Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors. PMID:27034984

  5. Mutations in chromatin machinery and pediatric high-grade glioma

    PubMed Central

    Lulla, Rishi R.; Saratsis, Amanda Muhs; Hashizume, Rintaro

    2016-01-01

    Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors. PMID:27034984

  6. Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

    PubMed

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

    2015-11-01

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

  7. Cellular and molecular etiology of hepatocyte injury in a murine model of environmentally induced liver abnormality

    PubMed Central

    Al-Griw, M.A.; Alghazeer, R.O.; Al-Azreg, S.A.; Bennour, E.M.

    2016-01-01

    Exposures to a wide variety of environmental substances are negatively associated with many biological cell systems both in humans and rodents. Trichloroethane (TCE), a ubiquitous environmental toxicant, is used in large quantities as a dissolvent, metal degreaser, chemical intermediate, and component of consumer products. This increases the likelihood of human exposure to these compounds through dermal, inhalation and oral routes. The present in vivo study was aimed to investigate the possible cellular and molecular etiology of liver abnormality induced by early exposure to TCE using a murine model. The results showed a significant increase in liver weight. Histopathological examination revealed a TCE-induced hepatotoxicity which appeared as heavily congested central vein and blood sinusoids as well as leukocytic infiltration. Mitotic figures and apoptotic changes such as chromatin condensation and nuclear fragments were also identified. Cell death analysis demonstrates hepatocellular apoptosis was evident in the treated mice compared to control. TCE was also found to induce oxidative stress as indicated by an increase in the levels of lipid peroxidation, an oxidative stress marker. There was also a significant decrease in the DNA content of the hepatocytes of the treated groups compared to control. Agarose gel electrophoresis also provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation in the liver cells, indicating oxidative stress as the cause of DNA damage. These results suggest the need for a complete risk assessment of any new chemical prior to its arrival into the consumer market. PMID:27800299

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

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

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

  9. The occurrence of period IV elements in dinoflagellate chromatin: an X-ray microanalytical study.

    PubMed

    Kearns, L P; Sigee, D C

    1980-12-01

    X-ray microanalysis of unfixed frozen-dried cryosections, and of unfixed cryosections collected by a sucrose drop technique, revealed the presence of Ca, Mn, Fe, Ni, Cu and Zn (Period IV, Periodic Table) in association with the permanently condensed chromosomes of Prorocentrum micans. All of these metals, with the exception of Mn, routinely occurred in the chromatin of cells that had been fixed in glutaraldehyde, ethanol-dehydrated, and resin-embedded. Their detection at high levels in chemically dehydrated chromatin of Prorocentrum micans, and 3 other dinoflagellate species cultured in the laboratory, suggests they are important as bound (insoluble) components of the chromatin. Detailed analysis of the fixed, ethanol-dehydrated material showed that for these particular cultures, each species had its own characteristic mass fraction pattern of chromatin-bound elements which differed markedly from that of the other species. Calculation of relative numbers of atoms from the mass fraction showed that for each species about 40-50 atoms of bound Period IV elements were present per 100 atoms of phosphorus. This is approximately equivalent to one Period IV atom per 2 DNA nucleotides or per base pair. Although considerable variation exists between species in terms of individual element levels, the total number of atoms of Period IV elements relative to phosphorus is remarkably constant. PMID:7194876

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Daban, Joan-Ramon

    2015-10-08

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

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

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

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

  14. The XXXXY Sex Chromosome Abnormality

    PubMed Central

    Barr, M. L.; Carr, D. H.; Pozsonyi, J.; Wilson, R. A.; Dunn, H. G.; Jacobson, T. S.; Miller, J. R.; Chown, B.

    1962-01-01

    The most common sex chromosome complex in sex chromatin-positive males with Klinefelter's syndrome is XXY. When the complex is XXYY or XXXY, the clinical findings do not seem to differ materially from those seen in XXY subjects, although more patients with these intersexual chromosome complements need to be studied to establish possible phenotypical expressions of the chromosomal variants. Two male children with an XXXXY sex chromosome abnormality are described. The data obtained from the study of these cases and five others described in the literature suggest that the XXXXY patient is likely to have congenital defects not usually seen in the common form of the Klinefelter syndrome. These include a triad of (1) skeletal anomalies (including radioulnar synostosis), (2) hypogenitalism (hypoplasia of penis and scrotum, incomplete descent of testes and defective prepubertal development of seminiferous tubules), and (3) greater risk of severe mental deficiency. That the conclusions are based on data from a small number of patients is emphasized, together with the need for a cytogenetic survey of a large control or unselected population. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10 PMID:13969480

  15. Chromatin Ring Formation at Plant Centromeres.

    PubMed

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

  16. Chromatin States Accurately Classify Cell Differentiation Stages

    PubMed Central

    Larson, Jessica L.; Yuan, Guo-Cheng

    2012-01-01

    Gene expression is controlled by the concerted interactions between transcription factors and chromatin regulators. While recent studies have identified global chromatin state changes across cell-types, it remains unclear to what extent these changes are co-regulated during cell-differentiation. Here we present a comprehensive computational analysis by assembling a large dataset containing genome-wide occupancy information of 5 histone modifications in 27 human cell lines (including 24 normal and 3 cancer cell lines) obtained from the public domain, followed by independent analysis at three different representations. We classified the differentiation stage of a cell-type based on its genome-wide pattern of chromatin states, and found that our method was able to identify normal cell lines with nearly 100% accuracy. We then applied our model to classify the cancer cell lines and found that each can be unequivocally classified as differentiated cells. The differences can be in part explained by the differential activities of three regulatory modules associated with embryonic stem cells. We also found that the “hotspot” genes, whose chromatin states change dynamically in accordance to the differentiation stage, are not randomly distributed across the genome but tend to be embedded in multi-gene chromatin domains, and that specialized gene clusters tend to be embedded in stably occupied domains. PMID:22363642

  17. Chromatin associations in Arabidopsis interphase nuclei

    PubMed Central

    Schubert, Veit; Rudnik, Radoslaw; Schubert, Ingo

    2014-01-01

    The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analyzed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fiber movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns. Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its 10 centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei. PMID:25431580

  18. Chromatin Ring Formation at Plant Centromeres

    PubMed Central

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

  19. Condensates in Jovian Atmospheres

    NASA Technical Reports Server (NTRS)

    West, R.

    1999-01-01

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

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

    SciTech Connect

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

    2010-11-11

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

  1. [Comparative characteristics of chromatin endonuclease fragments].

    PubMed

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

    1977-05-01

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

  2. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

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

    2012-01-01

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

  3. The Chromatin Fiber: Multiscale Problems and Approaches

    PubMed Central

    Ozer, Gungor; Luque, Antoni; Schlick, Tamar

    2015-01-01

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

  4. Nucleosome dynamics during chromatin remodeling in vivo

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    ABSTRACT Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation. PMID:26933790

  5. Functions of the Proteasome on Chromatin

    PubMed Central

    McCann, Tyler S.; Tansey, William P.

    2014-01-01

    The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome. PMID:25422899

  6. Nuclease digestion studies of chromatin structure

    SciTech Connect

    Deutsch, S.M.

    1987-01-01

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

  7. Open chromatin reveals the functional maize genome

    PubMed Central

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

    2016-01-01

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

  8. Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility.

    PubMed

    Lakpour, Niknam; Mahfouz, Reda Z; Akhondi, Mohammad Mehdi; Agarwal, Ashok; Kharrazi, Hadi; Zeraati, Hojjat; Amirjannati, Naser; Sadeghi, Mohammad Reza

    2012-10-01

    We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p < 0.001). Sperm chromatin damage was correlated with percentage of CMA(3)- positive sperm (r = 0.64, p < 0.001) and with sperm concentration (r = -0.36, p < 0.001), motility (r = -0.21, p < 0.05), and morphologically normal spermatozoa (r = -0.29, p < 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA(3)- positive sperm (p < 0.001) in ungrouped patients. It was related to both percentage of CMA(3)- positive sperm and serum DHEA in the MF group (p < 0.001 and p < 0.05, respectively). Sperm chromatin maturity assessed by CMA(3) test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.

  9. Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

    PubMed

    Sun, Zilong; Niu, Ruiyan; Wang, Bin; Wang, Jundong

    2014-06-01

    This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice.

  10. CHD chromatin remodelers and the transcription cycle.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

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

  11. Chromatin Control of Developmental Dynamics and Plasticity.

    PubMed

    Perino, Matteo; Veenstra, Gert Jan C

    2016-09-26

    Chromatin structure is intimately connected with gene expression and cell identity. Here we review recent advances in the field and discuss how establishment of cell identity during development is accompanied by large-scale remodeling of the epigenetic landscape and how this remodeling drives and supports lineage specification and maintenance. We discuss maternal control of the early embryonic epigenetic landscape, selective usage of enhancer clusters via 3D chromatin contacts leading to activation of transcription factor networks, and conserved regulation of developmental pathways by specific DNA demethylation of key regulatory regions. Together, these processes establish an epigenetic framework regulating different phases of embryonic development. PMID:27676434

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

    PubMed

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

    2015-12-24

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

  13. Probing chromatin landscape reveals roles of endocardial TBX20 in septation

    PubMed Central

    Boogerd, Cornelis J.; Aneas, Ivy; Sakabe, Noboru; Dirschinger, Ralph J.; Cheng, Quen J.; Chen, Ju; Nobrega, Marcelo A.; Evans, Sylvia M.

    2016-01-01

    Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20–binding site–dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation. PMID:27348591

  14. Histone H1 compacts DNA under force and during chromatin assembly.

    PubMed

    Xiao, Botao; Freedman, Benjamin S; Miller, Kelly E; Heald, Rebecca; Marko, John F

    2012-12-01

    Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly. PMID:23097493

  15. ATP-Dependent Chromatin Remodeling Complexes as Novel Targets for Cancer Therapy

    PubMed Central

    Mayes, Kimberly; Qiu, Zhijun; Alhazmi, Aiman; Landry, Joseph W.

    2016-01-01

    The progression to advanced stage cancer requires changes in many characteristics of a cell. These changes are usually initiated through spontaneous mutation. As a result of these mutations, gene expression is almost invariably altered allowing the cell to acquire tumor-promoting characteristics. These abnormal gene expression patterns are in part enabled by the posttranslational modification and remodeling of nucleosomes in chromatin. These chromatin modifications are established by a functionally diverse family of enzymes including histone and DNA-modifying complexes, histone deposition pathways, and chromatin remodeling complexes. Because the modifications these enzymes deposit are essential for maintaining tumor-promoting gene expression, they have recently attracted much interest as novel therapeutic targets. One class of enzyme that has not generated much interest is the chromatin remodeling complexes. In this review, we will present evidence from the literature that these enzymes have both causal and enabling roles in the transition to advanced stage cancers; as such, they should be seriously considered as high-value therapeutic targets. Previously published strategies for discovering small molecule regulators to these complexes are described. We close with thoughts on future research, the field should perform to further develop this potentially novel class of therapeutic target. PMID:24889532

  16. Restraint of angiogenesis by zinc finger transcription factor CTCF-dependent chromatin insulation

    PubMed Central

    Tang, Ming; Chen, Bo; Pardo, Carolina; Pampo, Christine; Chen, Jing; Lien, Ching-Ling; Wu, Lizi; Wang, Heiman; Yao, Kai; Oh, S. Paul; Seto, Edward; Smith, Lois E. H.; Siemann, Dietmar W.; Kladde, Michael P.; Cepko, Constance L.; Lu, Jianrong

    2011-01-01

    Angiogenesis is meticulously controlled by a fine balance between positive and negative regulatory activities. Vascular endothelial growth factor (VEGF) is a predominant angiogenic factor and its dosage is precisely regulated during normal vascular formation. In cancer, VEGF is commonly overproduced, resulting in abnormal neovascularization. VEGF is induced in response to various stimuli including hypoxia; however, very little is known about the mechanisms that confine its induction to ensure proper angiogenesis. Chromatin insulation is a key transcription mechanism that prevents promiscuous gene activation by interfering with the action of enhancers. Here we show that the chromatin insulator-binding factor CTCF binds to the proximal promoter of VEGF. Consistent with the enhancer-blocking mode of chromatin insulators, CTCF has little effect on basal expression of VEGF but specifically affects its activation by enhancers. CTCF knockdown cells are sensitized for induction of VEGF and exhibit elevated proangiogenic potential. Cancer-derived CTCF missense mutants are mostly defective in blocking enhancers at the VEGF locus. Moreover, during mouse retinal development, depletion of CTCF causes excess angiogenesis. Therefore, CTCF-mediated chromatin insulation acts as a crucial safeguard against hyperactivation of angiogenesis. PMID:21896759

  17. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... The appearance of normal teeth varies, especially the molars. ... conditions. Specific diseases can affect tooth shape, tooth ...

  18. Chromatin regulation: how complex does it get?

    PubMed

    Meier, Karin; Brehm, Alexander

    2014-11-01

    Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general. PMID:25482055

  19. The great repression: chromatin and cryptic transcription.

    PubMed

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

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

  20. Trivalent chromatin marks the way in.

    PubMed

    Hysolli, Eriona; Park, In-Hyun

    2013-11-01

    Recently in Cell, Wapinski et al. (2013) investigated the epigenetic mechanisms underlying the direct conversion of fibroblasts to induced neurons (iNs). They found that Ascl1 acts as a pioneer factor at neurogenic loci marked by a closed "trivalent" chromatin state in cells permissive to direct conversion, but not in restrictive cell types. PMID:24209756

  1. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

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

  2. Chemical biology: Chromatin chemistry goes cellular

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  3. Chromatin regulation: How complex does it get?

    PubMed Central

    Meier, Karin; Brehm, Alexander

    2014-01-01

    Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general. PMID:25482055

  4. Unraveling the mechanisms of chromatin fibril packaging.

    PubMed

    Gavrilov, Alexey A; Shevelyov, Yuri Y; Ulianov, Sergey V; Khrameeva, Ekaterina E; Kos, Pavel; Chertovich, Alexander; Razin, Sergey V

    2016-05-01

    Recent data indicate that eukaryotic chromosomes are organized into Topologically Associating Domains (TADs); however, the mechanisms underlying TAD formation remain obscure. Based on the results of Hi-C analysis performed on 4 Drosophila melanogaster cell lines, we have proposed that specific properties of nucleosomes in active and repressed chromatin play a key role in the formation of TADs. Our computer simulations showed that the ability of "inactive" nucleosomes to stick to each other and the lack of such ability in "active" nucleosomes is sufficient for spatial segregation of these types of chromatin, which is revealed in the Hi-C analysis as TAD/inter-TAD partitioning. However, some Drosophila and mammalian TADs contain both active and inactive chromatin, a fact that does not fit this model. Herein, we present additional arguments for the model by postulating that transcriptionally active chromatin is extruded on the surface of a TAD, and discuss the possible impact of this organization on the enhancer-promoter communication and on the segregation of TADs. PMID:27249516

  5. Interplay between mismatch repair and chromatin assembly

    PubMed Central

    Schöpf, Barbara; Bregenhorn, Stephanie; Quivy, Jean-Pierre; Kadyrov, Farid A.; Almouzni, Genevieve; Jiricny, Josef

    2012-01-01

    Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1. PMID:22232658

  6. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

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

  7. Epigenetic chromatin silencing: bistability and front propagation

    PubMed Central

    Sedighi, Mohammad; Sengupta, Anirvan M

    2008-01-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally. PMID:17991991

  8. Proceedings: Condenser technology conference

    SciTech Connect

    Tsou, J.L. ); Mussalli, Y.G. )

    1991-08-01

    Seam surface condenser and associated systems performance strongly affects availability and heat rate in nuclear and fossil power plants. Thirty-six papers presented at a 1990 conference discuss research results, industry experience, and case histories of condenser problems and solutions. This report contains papers on life extension, performance improvement, corrosion and failure analysis, fouling prevention, and recommendation for future R D. The information represents recent work on condenser problems and solutions to improve the procurement, operation, and maintenance functions of power plant personnel. Several key points follow: A nuclear and a fossil power plant report show that replacing titanium tube bundles improves condenser availability and performance. One paper reports 10 years of experience with enhanced heat transfer tubes in utility condensers. The newly developed enhanced condenser tubes could further improve condensing heat transfer. A new resistance summation method improves the accuracy of condenser performance prediction, especially for stainless steel and titanium tubed condensers. Several papers describe improved condenser fouling monitoring techniques, including a review of zebra mussel issues.

  9. Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle

    PubMed Central

    Marcos-Villar, Laura; Pazo, Alejandra

    2016-01-01

    ABSTRACT Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1–RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle. IMPORTANCE Although influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA

  10. Understanding RNA-Chromatin Interactions Using Chromatin Isolation by RNA Purification (ChIRP).

    PubMed

    Chu, Ci; Chang, Howard Y

    2016-01-01

    ChIRP is a novel and easy-to-use technique for studying long noncoding RNA (lncRNA)-chromatin interactions. RNA and chromatin are cross-linked in vivo using formaldehyde or glutaraldehyde, and purified using biotinylated antisense oligonucleotides that hybridize to the target RNA. Co-precipitated DNA is then purified and analyzed by quantitative PCR (qPCR) or high-throughput sequencing. PMID:27659979

  11. Direct chromatin PCR (DC-PCR): hypotonic conditions allow differentiation of chromatin states during thermal cycling.

    PubMed

    Vatolin, Sergei; Khan, Shahper N; Reu, Frederic J

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.

  12. Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

    PubMed Central

    Xue, John Z.; Woo, Eileen M.; Postow, Lisa; Chait, Brian T.; Funabiki, Hironori

    2013-01-01

    SUMMARY Nuclear shape and size vary between species, during development and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). While microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators. PMID:24075807

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

    PubMed

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

    2015-01-01

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

  14. Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae.

    PubMed

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J

    2014-04-01

    The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

  15. Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

    PubMed Central

    Rybaczek, Dorota; Musiałek, Marcelina Weronika; Balcerczyk, Aneta

    2015-01-01

    We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant–DSBs versus alkaline–DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD. PMID:26545248

  16. Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota; Musiałek, Marcelina Weronika; Balcerczyk, Aneta

    2015-01-01

    We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD. PMID:26545248

  17. Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota; Musiałek, Marcelina Weronika; Balcerczyk, Aneta

    2015-01-01

    We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

  18. Structurally abnormal human autosomes

    SciTech Connect

    1993-12-31

    Chapter 25, discusses structurally abnormal human autosomes. This discussion includes: structurally abnormal chromosomes, chromosomal polymorphisms, pericentric inversions, paracentric inversions, deletions or partial monosomies, cri du chat (cat cry) syndrome, ring chromosomes, insertions, duplication or pure partial trisomy and mosaicism. 71 refs., 8 figs.

  19. Morphological abnormalities among lampreys

    USGS Publications Warehouse

    Manion, Patrick J.

    1967-01-01

    The experimental control of the sea lamprey (Petromyzon marinus) in the Great Lakes has required the collection of thousands of lampreys. Representatives of each life stage of the four species of the Lake Superior basin were examined for structural abnormalities. The most common aberration was the presence of additional tails. The accessory tails were always postanal and smaller than the normal tail. The point of origin varied; the extra tails occurred on dorsal, ventral, or lateral surfaces. Some of the extra tails were misshaped and curled, but others were normal in shape and pigment pattern. Other abnormalities in larval sea lampreys were malformed or twisted tails and bodies. The cause of the structural abnormalities is unknown. The presence of extra caudal fins could be genetically controlled, or be due to partial amputation or injury followed by abnormal regeneration. Few if any lampreys with structural abnormalities live to sexual maturity.

  20. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

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

    2005-11-01

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

  1. Kinetochores and chromatin diminution in early embryos of Parascaris univalens

    PubMed Central

    1992-01-01

    In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization. PMID:1618905

  2. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

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

    2016-07-01

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses. PMID:27391925

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

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Sandhu, Tejdev; Nordlund, Thomas

    2004-11-01

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

  4. Histone H3.3 regulates dynamic chromatin states during spermatogenesis

    PubMed Central

    Yuen, Benjamin T. K.; Bush, Kelly M.; Barrilleaux, Bonnie L.; Cotterman, Rebecca; Knoepfler, Paul S.

    2014-01-01

    The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics. PMID:25142466

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

    PubMed

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

    2015-12-17

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

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

    PubMed

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

    2015-12-17

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

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

    PubMed

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

    2016-06-30

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

  8. Minireview: Conversing With Chromatin: The Language of Nuclear Receptors

    PubMed Central

    2014-01-01

    Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners. PMID:24196351

  9. Minireview: Conversing with chromatin: the language of nuclear receptors.

    PubMed

    Biddie, Simon C; John, Sam

    2014-01-01

    Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners. PMID:24196351

  10. Effect of rabbit age on sperm chromatin structure.

    PubMed

    Gogol, P; Bochenek, M; Smorag, Z

    2002-04-01

    The aim of this study was to determine the relationship between the age of male rabbits and the sperm chromatin structure. The studies involved the semen of New Zealand White rabbits between 5 and 28 months of age. A flow cytometry and sperm chromatin structure assay (SCSA) method was used to determine chromatin structure. The results of cytometric chromatin structure assay suggested a relatively high stability of sperm chromatin in the rabbit. Between 6 and 16 months of age, the mean percentage of sperm with damaged chromatin was the lowest and ranged from 1.7 to 2.4%. Decreased sperm chromatin stability was found in ejaculates taken from male rabbits less than 5 months and more than 20 months of age. PMID:11975746

  11. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

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

    2016-01-01

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

  12. Calorie restriction and the exercise of chromatin

    PubMed Central

    Vaquero, Alejandro; Reinberg, Danny

    2009-01-01

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

  13. Regulation of chromatin by histone modifications

    PubMed Central

    Bannister, Andrew J; Kouzarides, Tony

    2011-01-01

    Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place. PMID:21321607

  14. Abnormal uterine bleeding.

    PubMed

    Jennings, J C

    1995-11-01

    Physicians who care for female patients cannot avoid the frequent complaint of abnormal uterine bleeding. Knowledge of the disorders that cause this problem can prevent serious consequences in many patients and improve the quality of life for many others. The availability of noninvasive and minimally invasive diagnostic studies and minimally invasive surgical treatment has revolutionized management of abnormal uterine bleeding. Similar to any other disorder, the extent to which a physician manages abnormal uterine bleeding depends on his or her own level of comfort. When limitations of either diagnostic or therapeutic capability are encountered, consultation and referral should be used to the best interest of patients.

  15. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  16. Measure Guideline: Evaporative Condensers

    SciTech Connect

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

    2012-03-01

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

  17. Chromatin modifications remodel cardiac gene expression.

    PubMed

    Mathiyalagan, Prabhu; Keating, Samuel T; Du, Xiao-Jun; El-Osta, Assam

    2014-07-01

    Signalling and transcriptional control involve precise programmes of gene activation and suppression necessary for cardiovascular physiology. Deep sequencing of DNA-bound transcription factors reveals a remarkable complexity of co-activators or co-repressors that serve to alter chromatin modification and regulate gene expression. The regulated complexes characterized by genome-wide mapping implicate the recruitment and exchange of proteins with specific enzymatic activities that include roles for histone acetylation and methylation in key developmental programmes of the heart. As for transcriptional changes in response to pathological stress, co-regulatory complexes are also differentially utilized to regulate genes in cardiac disease. Members of the histone deacetylase (HDAC) family catalyse the removal of acetyl groups from proteins whose pharmacological inhibition has profound effects preventing heart failure. HDACs interact with a complex co-regulatory network of transcription factors, chromatin-remodelling complexes, and specific histone modifiers to regulate gene expression in the heart. For example, the histone methyltransferase (HMT), enhancer of zeste homolog 2 (Ezh2), is regulated by HDAC inhibition and associated with pathological cardiac hypertrophy. The challenge now is to target the activity of enzymes involved in protein modification to prevent or reverse the expression of genes implicated with cardiac hypertrophy. In this review, we discuss the role of HDACs and HMTs with a focus on chromatin modification and gene function as well as the clinical treatment of heart failure. PMID:24812277

  18. Geothermal steam condensate reinjection

    NASA Technical Reports Server (NTRS)

    Chasteen, A. J.

    1974-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

  2. "Jeopardy" in Abnormal Psychology.

    ERIC Educational Resources Information Center

    Keutzer, Carolin S.

    1993-01-01

    Describes the use of the board game, Jeopardy, in a college level abnormal psychology course. Finds increased student interaction and improved application of information. Reports generally favorable student evaluation of the technique. (CFR)

  3. Abnormal Uterine Bleeding

    MedlinePlus

    ... Abnormal uterine bleeding is any bleeding from the uterus (through your vagina) other than your normal monthly ... or fibroids (small and large growths) in the uterus can also cause bleeding. Rarely, a thyroid problem, ...

  4. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... as cancer of the uterus, cervix, or vagina • Polycystic ovary syndrome How is abnormal bleeding diagnosed? Your health care ... before the fetus can survive outside the uterus. Polycystic Ovary Syndrome: A condition characterized by two of the following ...

  5. Freeze-Tolerant Condensers

    NASA Technical Reports Server (NTRS)

    Crowley, Christopher J.; Elkouhk, Nabil

    2004-01-01

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

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

    SciTech Connect

    Terry, Samantha Y.A.

    2012-07-15

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

  7. SWI/SNF chromatin remodeling complexes and cancer.

    PubMed

    Biegel, Jaclyn A; Busse, Tracy M; Weissman, Bernard E

    2014-09-01

    The identification of mutations and deletions in the SMARCB1 locus in chromosome band 22q11.2 in pediatric rhabdoid tumors provided the first evidence for the involvement of the SWI/SNF chromatin remodeling complex in cancer. Over the last 15 years, alterations in more than 20 members of the complex have been reported in a variety of human tumors. These include germline mutations and copy number alterations in SMARCB1, SMARCA4, SMARCE1, and PBRM1 that predispose carriers to both benign and malignant neoplasms. Somatic mutations, structural abnormalities, or epigenetic modifications that lead to reduced or aberrant expression of complex members have now been reported in more than 20% of malignancies, including both solid tumors and hematologic disorders in both children and adults. In this review, we will highlight the role of SMARCB1 in cancer as a paradigm for other tumors with alterations in SWI/SNF complex members and demonstrate the broad spectrum of mutations observed in complex members in different tumor types.

  8. SWI/SNF chromatin remodeling and human malignancies.

    PubMed

    Masliah-Planchon, Julien; Bièche, Ivan; Guinebretière, Jean-Marc; Bourdeaut, Franck; Delattre, Olivier

    2015-01-01

    The SWI/SNF complexes, initially identified in yeast 20 years ago, are a family of multi-subunit complexes that use the energy of adenosine triphosphate (ATP) hydrolysis to remodel nucleosomes. Chromatin remodeling processes mediated by the SWI/SNF complexes are critical to the modulation of gene expression across a variety of cellular processes, including stemness, differentiation, and proliferation. The first evidence of the involvement of these complexes in carcinogenesis was provided by the identification of biallelic, truncating mutations of the SMARCB1 gene in malignant rhabdoid tumors, a highly aggressive childhood cancer. Subsequently, genome-wide sequencing technologies have identified mutations in genes encoding different subunits of the SWI/SNF complexes in a large number of tumors. SWI/SNF mutations, and the subsequent abnormal function of SWI/SNF complexes, are among the most frequent gene alterations in cancer. The mechanisms by which perturbation of the SWI/SNF complexes promote oncogenesis are not fully elucidated; however, alterations of SWI/SNF genes obviously play a major part in cancer development, progression, and/or resistance to therapy.

  9. Comparative analysis of ATRX, a chromatin remodeling protein.

    PubMed

    Park, Daniel J; Pask, Andrew J; Huynh, Kim; Renfree, Marilyn B; Harley, Vincent R; Graves, Jennifer A Marshall

    2004-09-15

    The ATRX protein, associated with X-linked alpha-thalassaemia, mental retardation and developmental abnormalities including genital dysgenesis, has been proposed to function as a global transcriptional regulator within a multi-protein complex. However, an understanding of the composition and mechanics of this machinery has remained elusive. We applied inter-specific comparative analysis to identify conserved elements which may be involved in regulating the conformation of chromatin. As part of this study, we cloned and sequenced the entire translatable coding region (7.4 kb) of the ATRX gene from a model marsupial (tammar wallaby, Macropus eugenii). We identify an ATRX ancestral core, conserved between plants, fish and mammals, comprising the cysteine-rich and SWI2/SNF2 helicase-like regions and protein interaction domains. Our data are consistent with the model of the cysteine-rich region as a DNA-binding zinc finger adjacent to a protein-binding (plant homeodomain-like) domain. Alignment of vertebrate ATRX sequences highlights other conserved elements, including a negatively charged mammalian sequence which we propose to be involved in binding of positively charged histone tails.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Global Chromatin Domain Organization of the Drosophila Genome

    PubMed Central

    de Wit, Elzo; Braunschweig, Ulrich; Greil, Frauke; Bussemaker, Harmen J.; van Steensel, Bas

    2008-01-01

    In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution. PMID:18369463

  14. Sedimentary condensation and authigenesis

    NASA Astrophysics Data System (ADS)

    Föllmi, Karl

    2016-04-01

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

  15. Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution

    SciTech Connect

    Aubele, M.; Juetting, U.R.; Rodenacker, K.; Gais, P.; Burger, G.; Hacker-Klom, U. )

    1990-01-01

    Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.

  16. Electrolyte vapor condenser

    DOEpatents

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

    1983-01-01

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

  17. Electrolyte vapor condenser

    DOEpatents

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

    1983-02-08

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

  18. Let dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions

    NASA Astrophysics Data System (ADS)

    Suzuki, M.; Watanabe, M.; Kanai, T.; Kase, Y.; Yatagai, F.; Kato, T.; Matsubara, S.

    We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/mum were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/mum. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all the mutants induced by 124 keV/mum carbon-ion beams showed deletion of the entire gene, while all mutants induced by 230keV/mum carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.

  19. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

  1. Ghost condensate busting

    SciTech Connect

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

    2008-09-15

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

  2. THE COLOR GLASS CONDENSATE.

    SciTech Connect

    MCLERRAN,L.

    2001-08-26

    The Color Glass Condensate is a state of high density gluonic matter which controls the high energy limit of hadronic interactions. Its properties are important for the initial conditions for matter produced at RHIC.

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

    SciTech Connect

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

    2008-06-10

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

  4. Measure Guideline: Evaporative Condensers

    SciTech Connect

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

    2012-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  6. Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

    PubMed

    Tong, Kevin; Skibbens, Robert V

    2015-06-01

    Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

  7. [Hair shaft abnormalities].

    PubMed

    Itin, P H; Düggelin, M

    2002-05-01

    Hair shaft disorders may lead to brittleness and uncombable hair. In general the hair feels dry and lusterless. Hair shaft abnormalities may occur as localized or generalized disorders. Genetic predisposition or exogenous factors are able to produce and maintain hair shaft abnormalities. In addition to an extensive history and physical examination the most important diagnostic examination to analyze a hair shaft problem is light microscopy. Therapy of hair shaft disorders should focus to the cause. In addition, minimizing traumatic influences to hair shafts, such as dry hair with an electric dryer, permanent waves and dyes is important. A short hair style is more suitable for such patients with hair shaft disorders.

  8. Condensate dark matter stars

    SciTech Connect

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

    2012-06-01

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

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

    PubMed

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

    2015-10-30

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

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

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

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

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

    PubMed

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

    2015-10-30

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

  12. Isolation of In Vivo SUMOylated Chromatin-Bound Proteins.

    PubMed

    Bawa-Khalfe, Tasneem

    2016-01-01

    SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems. PMID:27631808

  13. Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

    PubMed Central

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

    2014-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

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

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

    PubMed

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

    2015-03-01

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

  16. The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

    PubMed Central

    2010-01-01

    Background Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary complex with Ran, but

  17. Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling

    PubMed Central

    Riparbelli, Maria Giovanna; Giordano, Rosanna; Ueyama, Morio; Callaini, Giuliano

    2012-01-01

    Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon. PMID:22291901

  18. Aberrant T cell ERK pathway signaling and chromatin structure in lupus

    PubMed Central

    Gorelik, Gabriela; Richardson, Bruce

    2009-01-01

    Human systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies to nuclear components with subsequent immune complex formation and deposition in multiple organs. A combination of genetic and environmental factors is required for disease development, but how the environment interacts with the immune system in genetically predisposed hosts to cause lupus is unclear. Recent evidence suggests that environmental agents may alter T cell chromatin structure and gene expression through effects on DNA methylation, a repressive epigenetic mechanism promoting chromatin inactivation, to cause lupus in people with the appropriate genetic background. DNA methylation is regulated by ERK pathway signaling, and abnormalities in ERK pathway signaling may contribute to immune dysfunction in lupus through epigenetic effects on gene expression. This article reviews current evidence for epigenetic abnormalities, and in particular DNA demethylation, in the pathogenesis of idiopathic and some forms of drug induced lupus, and how impaired ERK pathway signaling may contribute to the development of human lupus through effects on T cell DNA methylation. PMID:18723128

  19. Facioscapulohumeral muscular dystrophy: consequences of chromatin relaxation

    PubMed Central

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

    2013-01-01

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

  20. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

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

    2011-11-04

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

  1. Condensed Matter Nuclear Science

    NASA Astrophysics Data System (ADS)

    Takahashi, Akito; Ota, Ken-Ichiro; Iwamura, Yashuhiro

    Preface -- 1. General. Progress in condensed matter nuclear science / A. Takahashi. Summary of ICCF-12 / X. Z. Li. Overview of light water/hydrogen-based low-energy nuclear reactions / G. H. Miley and P. J. Shrestha -- 2. Excess heat and He detection. Development of "DS-reactor" as the practical reactor of "cold fusion" based on the "DS-cell" with "DS-cathode" / Y. Arata and Y.-C. Zhang. Progress in excess of power experiments with electrochemical loading of deuterium in palladium / V. Violante ... [et al.]. Anomalous energy generation during conventional electrolysis / T. Mizuno and Y. Toriyabe. "Excess heat" induced by deuterium flux in palladium film / B. Liu ... [et al.]. Abnormal excess heat observed during Mizuno-type experiments / J.-F. Fauvarque, P. P. Clauzon and G. J.-M. Lallevé. Seebeck envelope calorimetry with a Pd|D[symbol]O + H[symbol]SO[symbol] electrolytic cell / W.-S. Zhang, J. Dash and Q. Wang. Observation and investigation of nuclear fusion and self-induced electric discharges in liquids / A. I. Koldamasov ... [et al.]. Description of a sensitive seebeck calorimeter used for cold fusion studies / E. Storms. Some recent results at ENEA / M. Apicella ... [et al.]. Heat measurement during plasma electrolysis / K. Iizumi ... [et al.]. Effect of an additive on thermal output during electrolysis of heavy water with a palladium cathode / Q. Wang and J. Dash. Thermal analysis of calorimetric systems / L. D'Aulerio ... [et al.]. Surface plasmons and low-energy nuclear reactions triggering / E. Castagna ... [et al.]. Production method for violent TCB jet plasma from cavity / F. Amini. New results and an ongoing excess heat controversy / L. Kowalski ... [et al.] -- 3. Transmutation. Observation of surface distribution of products by X-ray fluorescence spectrometry during D[symbol] gas permeation through Pd Complexes / Y. Iwamura ... [et al.]. Discharge experiment using Pd/CaO/Pd multi-layered cathode / S. Narita ... [et al.]. Producing transmutation

  2. Condensed Matter Nuclear Science

    NASA Astrophysics Data System (ADS)

    Takahashi, Akito; Ota, Ken-Ichiro; Iwamura, Yashuhiro

    Preface -- 1. General. Progress in condensed matter nuclear science / A. Takahashi. Summary of ICCF-12 / X. Z. Li. Overview of light water/hydrogen-based low-energy nuclear reactions / G. H. Miley and P. J. Shrestha -- 2. Excess heat and He detection. Development of "DS-reactor" as the practical reactor of "cold fusion" based on the "DS-cell" with "DS-cathode" / Y. Arata and Y.-C. Zhang. Progress in excess of power experiments with electrochemical loading of deuterium in palladium / V. Violante ... [et al.]. Anomalous energy generation during conventional electrolysis / T. Mizuno and Y. Toriyabe. "Excess heat" induced by deuterium flux in palladium film / B. Liu ... [et al.]. Abnormal excess heat observed during Mizuno-type experiments / J.-F. Fauvarque, P. P. Clauzon and G. J.-M. Lallevé. Seebeck envelope calorimetry with a Pd|D[symbol]O + H[symbol]SO[symbol] electrolytic cell / W.-S. Zhang, J. Dash and Q. Wang. Observation and investigation of nuclear fusion and self-induced electric discharges in liquids / A. I. Koldamasov ... [et al.]. Description of a sensitive seebeck calorimeter used for cold fusion studies / E. Storms. Some recent results at ENEA / M. Apicella ... [et al.]. Heat measurement during plasma electrolysis / K. Iizumi ... [et al.]. Effect of an additive on thermal output during electrolysis of heavy water with a palladium cathode / Q. Wang and J. Dash. Thermal analysis of calorimetric systems / L. D'Aulerio ... [et al.]. Surface plasmons and low-energy nuclear reactions triggering / E. Castagna ... [et al.]. Production method for violent TCB jet plasma from cavity / F. Amini. New results and an ongoing excess heat controversy / L. Kowalski ... [et al.] -- 3. Transmutation. Observation of surface distribution of products by X-ray fluorescence spectrometry during D[symbol] gas permeation through Pd Complexes / Y. Iwamura ... [et al.]. Discharge experiment using Pd/CaO/Pd multi-layered cathode / S. Narita ... [et al.]. Producing transmutation

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

    PubMed

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

    2016-07-01

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

  4. Brd4 shields chromatin from ATM kinase signaling storms.

    PubMed

    Choi, Serah; Bakkenist, Christopher J

    2013-09-17

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

  5. A Broad Set of Chromatin Factors Influences Splicing

    PubMed Central

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

    2016-01-01

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

  6. Biochemical analysis of chromatin containing recombinant Drosophila core histones.

    PubMed

    Levenstein, Mark E; Kadonaga, James T

    2002-03-01

    To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones. To this end, we synthesized the Drosophila core histones in Escherichia coli. The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues. The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP. With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones. We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones. In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template. Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications.

  7. Brd4 Shields Chromatin from ATM Kinase Signaling Storms

    PubMed Central

    Choi, Serah; Bakkenist, Christopher J.

    2014-01-01

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

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

    PubMed

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

    2015-11-20

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

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

    PubMed

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

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. PMID:26381410

  10. Diagnostic cellular abnormalities in neoplastic and non-neoplastic lesions of the epidermis: a morphological and statistical study

    PubMed Central

    Malhotra, Saurabh; Kazlouskaya, Viktoryia; Andres, Christian; Gui, Jiang; Elston, Dirk

    2013-01-01

    Background Distinguishing cellular abnormalities in reactive and malignant lesions is challenging. We compared the incidence and severity of cytological abnormalities in malignant/premalignant and benign epidermal lesions. Methods One hundred fifty-two biopsies representing 69 malignant/premalignant squamous lesions and 83 benign conditions were studied. Cytological features, including nuclear hyperchromasia, nuclear overlap (crowding), irregular nuclei, high nuclear/cytoplasmic (N/C) ratio, conspicuous nucleoli, delicate inconspicuous nucleoli, clumped chromatin, pleomorphic parakeratosis, normal and abnormal mitotic figures and necrotic keratinocytes, were evaluated and graded. Statistical analysis was performed. Results Irregular nuclei, increased N/C ratio, conspicuous single prominent nucleoli, nuclear overlap (crowding), pleomorphic parakeratosis, nuclear hyperchromasia, necrotic keratinocytes, normal and abnormal mitotic figures and coarse chromatin were seen more frequently in malignant neoplasms (p < 0.05). Abnormal mitotic figures, although uncommon (20.3%), were only noted in the malignant/premalignant group. Certain cytological features were common among both malignant and benign lesions, suggesting that they are of little value. Conclusion In the setting of an atypical cutaneous squamous proliferation, nuclear irregularity, increased N/C ratio, conspicuous nucleoli, crowding and hyperchromasia are the most useful indicators of malignancy. In contrast, mitotic figures, necrotic cells and coarse chromatin are less useful. The presence of abnormal mitotic figures is very helpful when present; however, their overall rarity limits their utility. PMID:23398548

  11. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation

    PubMed Central

    Nguyen, Carvell T.; Gonzales, Felicidad A.; Jones, Peter A.

    2001-01-01

    Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands. PMID:11713309

  12. ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells

    PubMed Central

    Saladi, Srinivas Vinod

    2010-01-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure. PMID:20148317

  13. Nonhistone Proteins Control Gene Expression in Reconstituted Chromatin

    PubMed Central

    Barrett, T.; Maryanka, D.; Hamlyn, P. H.; Gould, H. J.

    1974-01-01

    Chromatin was reconstituted from the purified DNA and histones of chicken erythrocytes and the nonhistone proteins of either chicken reticulocytes or chicken liver. Reconstituted chromatins, native chicken reticulocyte chromatin, and free DNA were transcribed with Escherichia coli RNA polymerase and the concentrations of globin-specific sequences in the RNA products were measured by hybridization with [3H]DNA complementary to chicken globin messenger RNA. Reticulocyte, but not liver, nonhistone proteins were shown to activate the globin genes in reconstituted erythrocyte chromatin. The transcripts of native and reconstituted chromatins were indistinguishable in respect of both the total yield of the RNA and the fractional yield of globin-specific sequences. Images PMID:4140516

  14. Silent chromatin at the middle and ends: lessons from yeasts

    PubMed Central

    Bühler, Marc; Gasser, Susan M

    2009-01-01

    Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species. PMID:19629038

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2003-10-24

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

  17. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-01-01

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

  18. Morphological abnormalities in elasmobranchs.

    PubMed

    Moore, A B M

    2015-08-01

    A total of 10 abnormal free-swimming (i.e., post-birth) elasmobranchs are reported from The (Persian-Arabian) Gulf, encompassing five species and including deformed heads, snouts, caudal fins and claspers. The complete absence of pelvic fins in a milk shark Rhizoprionodon acutus may be the first record in any elasmobranch. Possible causes, including the extreme environmental conditions and the high level of anthropogenic pollution particular to The Gulf, are briefly discussed.

  19. Chromosome abnormalities in glioma

    SciTech Connect

    Li, Y.S.; Ramsay, D.A.; Fan, Y.S.

    1994-09-01

    Cytogenetic studies were performed in 25 patients with gliomas. An interesting finding was a seemingly identical abnormality, an extra band on the tip of the short arm of chromosome 1, add(1)(p36), in two cases. The abnormality was present in all cells from a patient with a glioblastoma and in 27% of the tumor cells from a patient with a recurrent irradiated anaplastic astrocytoma; in the latter case, 7 unrelated abnormal clones were identified except 4 of those clones shared a common change, -Y. Three similar cases have been described previously. In a patient with pleomorphic astrocytoma, the band 1q42 in both homologues of chromosome 1 was involved in two different rearrangements. A review of the literature revealed that deletion of the long arm of chromosome 1 including 1q42 often occurs in glioma. This may indicate a possible tumor suppressor gene in this region. Cytogenetic follow-up studies were carried out in two patients and emergence of unrelated clones were noted in both. A total of 124 clonal breakpoints were identified in the 25 patients. The breakpoints which occurred three times or more were: 1p36, 1p22, 1q21, 1q25, 3q21, 7q32, 8q22, 9q22, 16q22, and 22q13.

  20. [Congenital foot abnormalities].

    PubMed

    Delpont, M; Lafosse, T; Bachy, M; Mary, P; Alves, A; Vialle, R

    2015-03-01

    The foot may be the site of birth defects. These abnormalities are sometimes suspected prenatally. Final diagnosis depends on clinical examination at birth. These deformations can be simple malpositions: metatarsus adductus, talipes calcaneovalgus and pes supinatus. The prognosis is excellent spontaneously or with a simple orthopedic treatment. Surgery remains outstanding. The use of a pediatric orthopedist will be considered if malposition does not relax after several weeks. Malformations (clubfoot, vertical talus and skew foot) require specialized care early. Clubfoot is characterized by an equine and varus hindfoot, an adducted and supine forefoot, not reducible. Vertical talus combines equine hindfoot and dorsiflexion of the forefoot, which is performed in the midfoot instead of the ankle. Skew foot is suspected when a metatarsus adductus is resistant to conservative treatment. Early treatment is primarily orthopedic at birth. Surgical treatment begins to be considered after walking age. Keep in mind that an abnormality of the foot may be associated with other conditions: malposition with congenital hip, malformations with syndromes, neurological and genetic abnormalities. PMID:25524290

  1. Chromatin insulators: lessons from the fly.

    PubMed

    Gurudatta, B V; Corces, Victor G

    2009-07-01

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

  2. Protein tagging for chromatin immunoprecipitation from Arabidopsis.

    PubMed

    de Folter, Stefan

    2011-01-01

    A powerful method to identify binding sites in target genes is chromatin immunoprecipitation (ChIP), which allows the purification of in vivo formed complexes of a DNA-binding protein and associated DNA. Briefly, the method involves the fixation of plant tissue and the isolation of the total protein-DNA mixture, followed by an immunoprecipitation step with an antibody directed against the protein of interest and, subsequently, the DNA can be purified. Finally, the DNA can be analyzed by PCR for the enrichment of specific regions. A drawback of ChIP is that for each protein another antibody is needed. To overcome this, a generic strategy is possible using tags fused to the protein of interest. In this case, only antibody is needed against the tag. This protocol describes the tagging of proteins and how to perform ChIP. PMID:20931382

  3. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

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

  4. Visualizing Long Noncoding RNAs on Chromatin

    PubMed Central

    Hinten, Michael; Maclary, Emily; Gayen, Srimonta; Harris, Clair; Kalantry, Sundeep

    2016-01-01

    Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences within single cells. For example, RNA FISH provides information on both the expression level and localization of RNA transcripts and, when combined with detection of associated proteins and chromatin modifications, can lend essential insights into long noncoding RNA (lncRNA) function. Epigenetic effects have been postulated for many lncRNAs, but shown for only a few. Advances in in situ techniques and microscopy, however, now allow for visualization of lncRNAs that are expressed at very low levels or are not very stable. FISH-based detections of RNA and DNA coupled with immunological staining of proteins/histone modifications offer the possibility to connect lncRNAs to epigenetic effects. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at a high level of sensitivity using conventional fluorescence microscopy. PMID:26721489

  5. Keeping condensers clean

    SciTech Connect

    Wicker, K.

    2006-04-15

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

  6. Chromatin changes predict recurrence after radical prostatectomy

    PubMed Central

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

    2016-01-01

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

  7. Abnormal pressures as hydrodynamic phenomena

    USGS Publications Warehouse

    Neuzil, C.E.

    1995-01-01

    So-called abnormal pressures, subsurface fluid pressures significantly higher or lower than hydrostatic, have excited speculation about their origin since subsurface exploration first encountered them. Two distinct conceptual models for abnormal pressures have gained currency among earth scientists. The static model sees abnormal pressures generally as relict features preserved by a virtual absence of fluid flow over geologic time. The hydrodynamic model instead envisions abnormal pressures as phenomena in which flow usually plays an important role. This paper develops the theoretical framework for abnormal pressures as hydrodynamic phenomena, shows that it explains the manifold occurrences of abnormal pressures, and examines the implications of this approach. -from Author

  8. Simple Simulations of DNA Condensation

    SciTech Connect

    STEVENS,MARK J.

    2000-07-12

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

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

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

    Detail of Bright Angel stone vault, containing condenser, Hoffman condensation pump, Jennings vacuum heating pump, and misc. pipes and valves. - Grand Canyon Village Utilities, Grand Canyon National Park, Grand Canyon Village, Coconino County, AZ

  10. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

    PubMed Central

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G.; Becker, Jörg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G.

    2016-01-01

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I. PMID:27507044

  11. A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit.

    PubMed

    Blobel, Gerd A; Kadauke, Stephan; Wang, Eric; Lau, Alan W; Zuber, Johannes; Chou, Margaret M; Vakoc, Christopher R

    2009-12-25

    Mixed lineage leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M phase of the cell cycle. Unlike other chromatin-modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation, which may facilitate inheritance of active gene expression states during cell division. PMID:20064463

  12. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling.

    PubMed

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G; Becker, Jörg D; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G

    2016-08-10

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.

  13. Feeling Abnormal: Simulation of Deviancy in Abnormal and Exceptionality Courses.

    ERIC Educational Resources Information Center

    Fernald, Charles D.

    1980-01-01

    Describes activity in which student in abnormal psychology and psychology of exceptional children classes personally experience being judged abnormal. The experience allows the students to remember relevant research, become sensitized to the feelings of individuals classified as deviant, and use caution in classifying individuals as abnormal.…

  14. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-01

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

  15. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability

    PubMed Central

    Li, Lei; Shi, Lan; Yang, Shangda; Yan, Ruorong; Zhang, Di; Yang, Jianguo; He, Lin; Li, Wanjin; Yi, Xia; Sun, Luyang; Liang, Jing; Cheng, Zhongyi; Shi, Lei; Shang, Yongfeng; Yu, Wenhua

    2016-01-01

    Although SIRT7 is a member of sirtuin family proteins that are described as NAD+-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD+-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7. PMID:27436229

  16. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability.

    PubMed

    Li, Lei; Shi, Lan; Yang, Shangda; Yan, Ruorong; Zhang, Di; Yang, Jianguo; He, Lin; Li, Wanjin; Yi, Xia; Sun, Luyang; Liang, Jing; Cheng, Zhongyi; Shi, Lei; Shang, Yongfeng; Yu, Wenhua

    2016-01-01

    Although SIRT7 is a member of sirtuin family proteins that are described as NAD(+)-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD(+)-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7. PMID:27436229

  17. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-01

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism. PMID:27108550

  18. Condensate removal device

    DOEpatents

    Maddox, James W.; Berger, David D.

    1984-01-01

    A condensate removal device is disclosed which incorporates a strainer in unit with an orifice. The strainer is cylindrical with its longitudinal axis transverse to that of the vapor conduit in which it is mounted. The orifice is positioned inside the strainer proximate the end which is remoter from the vapor conduit.

  19. Inflation from gravitino condensates

    NASA Astrophysics Data System (ADS)

    Mavromatos, Nick E.

    2015-07-01

    We review work on the formation of gravitino condensates via the super-Higgs effect in the early Universe. This is a scenario for both inflating the early universe and breaking local supersymmetry(supergravity), entirely independent of any coupling to external matter. The goldstino mode associated with the breaking of (global) supersymmetry is “eaten” by the gravitino field, which becomes massive (via its own vacuum condensation) and breaks supergravity dynamically. The most natural association of gravitino condensates with inflation proceeds in an indirect way, via a Starobinsky-type inflation, in the massive gravitino phase. This inflationary phase is associated with scalar modes hidden in the higher order curvature corrections of the effective action arising from integrating out massive gravitino degrees of freedom. The scenario is in agreement with Planck data phenomenology in a natural and phenomenologically-relevant range of parameters, namely Grand-Unified-Theory values for the supersymmetry breaking energy scale and dynamically-induced gravitino mass. A hill-top inflation, on the other hand, which could also occur in the model, whereby the role of the inflaton field is played by the gravitino condensate itself, would require significant fine tuning in the inflaton's wave function renormalisation and thus may be discarded on naturalness grounds.

  20. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  1. Exercises to Improve Gait Abnormalities

    MedlinePlus

    ... Home About iChip Articles Directories Videos Resources Contact Exercises to Improve Gait Abnormalities Home » Article Categories » Exercise and Fitness Font Size: A A A A Exercises to Improve Gait Abnormalities Next Page The manner ...

  2. Abnormal ionization in sonoluminescence

    NASA Astrophysics Data System (ADS)

    Zhang, Wen-Juan; An, Yu

    2015-04-01

    Sonoluminescence is a complex phenomenon, the mechanism of which remains unclear. The present study reveals that an abnormal ionization process is likely to be present in the sonoluminescing bubble. To fit the experimental data of previous studies, we assume that the ionization energies of the molecules and atoms in the bubble decrease as the gas density increases and that the decrease of the ionization energy reaches about 60%-70% as the bubble flashes, which is difficult to explain by using previous models. Project supported by the Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20120002110031) and the National Natural Science Foundation of China (Grant No. 11334005).

  3. Fractal Characterization of Chromatin Decompaction in Live Cells.

    PubMed

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

    2015-12-01

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

  4. Abnormal hematological indices in cirrhosis

    PubMed Central

    Qamar, Amir A; Grace, Norman D

    2009-01-01

    Abnormalities in hematological indices are frequently encountered in cirrhosis. Multiple causes contribute to the occurrence of hematological abnormalities. Recent studies suggest that the presence of hematological cytopenias is associated with a poor prognosis in cirrhosis. The present article reviews the pathogenesis, incidence, prevalence, clinical significance and treatment of abnormal hematological indices in cirrhosis. PMID:19543577

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

    PubMed

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

    2015-02-18

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

  6. Nucleosome positioning and composition modulate in silico chromatin flexibility

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  7. Nucleosome positioning and composition modulate in silico chromatin flexibility

    PubMed Central

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

    2015-01-01

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

  8. Spatially confined folding of chromatin in the interphase nucleus

    PubMed Central

    Mateos-Langerak, Julio; Bohn, Manfred; de Leeuw, Wim; Giromus, Osdilly; Manders, Erik M. M.; Verschure, Pernette J.; Indemans, Mireille H. G.; Gierman, Hinco J.; Heermann, Dieter W.; van Driel, Roel; Goetze, Sandra

    2009-01-01

    Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10–30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin–chromatin interactions and determine the 3-dimensional organization of the chromatin fiber. PMID:19234129

  9. Nanoscale squeezing in elastomeric nanochannels for single chromatin linearization

    PubMed Central

    Matsuoka, Toshiki; Kim, Byoung Choul; Huang, Jiexi; Douville, Nicholas Joseph; Thouless, M.D.; Takayama, Shuichi

    2012-01-01

    This paper describes a novel nanofluidic phenomenon where untethered DNA and chromatin are linearized by rapidly narrowing an elastomeric nanochannel filled with solutions of the biopolymers. This nanoscale squeezing procedure generates hydrodynamic flows while also confining the biopolymers into smaller and smaller volumes. The unique features of this technique enable full linearization then trapping of biopolymers such as DNA. The versatility of the method is also demonstrated by analysis of chromatin stretchability and mapping of histone states using single strands of chromatin. PMID:23186544

  10. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  11. Stress-induced structural changes in plant chromatin.

    PubMed

    Probst, Aline V; Mittelsten Scheid, Ortrun

    2015-10-01

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

  12. Chromatin regulation at the frontier of synthetic biology

    PubMed Central

    Keung, Albert J.; Joung, J. Keith; Khalil, Ahmad S.; Collins, James J.

    2016-01-01

    As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including `epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication. PMID:25668787

  13. Spirometric abnormalities among welders

    SciTech Connect

    Rastogi, S.K.; Gupta, B.N.; Husain, T.; Mathur, N.; Srivastava, S. )

    1991-10-01

    A group of manual welders age group 13-60 years having a mean exposure period of 12.4 {plus minus} 1.12 years were subjected to spirometry to evaluate the prevalence of spirometric abnormalities. The welders showed a significantly higher prevalence of respiratory impairment than that observed among the unexposed controls as a result of exposure to welding gases which comprised fine particles of lead, zinc, chromium, and manganese. This occurred despite the lower concentration of the pollutants at the work place. In the expose group, the smoking welders showed a prevalence of respiratory impairment significantly higher than that observed in the nonsmoking welders. The results of the pulmonary function tests showed a predominantly restrictive type of pulmonary impairment followed by a mixed ventilatory defect among the welders. The effect of age on pulmonary impairment was not discernible. Welders exposed for over 10 years showed a prevalence of respiratory abnormalities significantly higher than those exposed for less than 10 years. Smoking also had a contributory role.

  14. [Diagnosis of MDS: morphology, chromosome abnormalities and genetic mutations].

    PubMed

    Hata, Tomoko

    2015-10-01

    Myelodysplastic syndromes (MDS) are a group of hematological neoplasms associated with ineffective hematopoiesis and that can transform into acute leukemia. The clinical classification of MDS which is defined by cytopenia, the rate of blasts in peripheral blood and bone marrow, dysplasia, and chromosomal abnormalities, has undergone continuous revision. To increase the accuracy of dysplastic evaluation, IWGM-MDS and the Research Committee for Idiopathic Hematopoietic Disorders, Ministry of Health, Labour and Welfare, Japan have proposed a quantitative and qualitative definition of dysplasia. Recently, refining the definition of dysgranulopoiesis was proposed by IWGM-MDS. Neutrophils with abnormal clumping of chromatin, and harboring more than 4 nuclear projections, were recognized as dysplastic features. At present, karyotypic abnormalities are detected in approximately 50% of de novo MDS and these remain the most critical prognostic factor. In the new cytogenetic scoring system, cytogenetic abnormalities were classified into five prognostic subgroups. This new classification was adopted by the revised IPSS. Approximately 80% to 90% of MDS patients have detectable mutations by whole-exon sequencing or whole genome sequencing. Many genetic mutations had biological and prognostic significance. It is important to further understand the utility of this factor in determining prognosis and in selecting among therapeutic options. PMID:26458436

  15. Photon condensation: A new paradigm for Bose-Einstein condensation

    NASA Astrophysics Data System (ADS)

    Rajan, Renju; Ramesh Babu, P.; Senthilnathan, K.

    2016-10-01

    Bose-Einstein condensation is a state of matter known to be responsible for peculiar properties exhibited by superfluid Helium-4 and superconductors. Bose-Einstein condensate (BEC) in its pure form is realizable with alkali atoms under ultra-cold temperatures. In this paper, we review the experimental scheme that demonstrates the atomic Bose-Einstein condensate. We also elaborate on the theoretical framework for atomic Bose-Einstein condensation, which includes statistical mechanics and the Gross-Pitaevskii equation. As an extension, we discuss Bose-Einstein condensation of photons realized in a fluorescent dye filled optical microcavity. We analyze this phenomenon based on the generalized Planck's law in statistical mechanics. Further, a comparison is made between photon condensate and laser. We describe how photon condensate may be a possible alternative for lasers since it does not require an energy consuming population inversion process.

  16. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

    PubMed Central

    Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

    2015-01-01

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

  17. Centromeric chromatin and its dynamics in plants.

    PubMed

    Lermontova, Inna; Sandmann, Michael; Mascher, Martin; Schmit, Anne-Catherine; Chabouté, Marie-Edith

    2015-07-01

    Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented.

  18. Chromatin immunoprecipitation analysis of Xenopus embryos.

    PubMed

    Akkers, Robert C; Jacobi, Ulrike G; Veenstra, Gert Jan C

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the epitope is associated with particular genomic regions can be obtained by quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or deep sequencing (ChIP-seq). ChIP can be used for molecular and quantitative analyses of histone modifications, transcription factors, and elongating RNA polymerase II at specific loci. It can also be applied to assess the cellular state of transcriptional activation or repression as a predictor of the cells' capabilities and potential. Another possibility is to employ ChIP to characterize genomes, as histone modifications and binding events occur at specific and highly characteristic genomic elements and locations. This chapter provides a step-by-step protocol of ChIP using early Xenopus embryos and discusses potential pitfalls and other issues relevant for successful probing of protein-genome interactions by ChIP-qPCR and ChIP-seq. PMID:22956095

  19. Gravity triggered neutrino condensates

    SciTech Connect

    Barenboim, Gabriela

    2010-11-01

    In this work we use the Schwinger-Dyson equations to study the possibility that an enhanced gravitational attraction triggers the formation of a right-handed neutrino condensate, inducing dynamical symmetry breaking and generating a Majorana mass for the right-handed neutrino at a scale appropriate for the seesaw mechanism. The composite field formed by the condensate phase could drive an early epoch of inflation. We find that to the lowest order, the theory does not allow dynamical symmetry breaking. Nevertheless, thanks to the large number of matter fields in the model, the suppression by additional powers in G of higher order terms can be compensated, boosting them up to their lowest order counterparts. This way chiral symmetry can be broken dynamically and the infrared mass generated turns out to be in the expected range for a successful seesaw scenario.

  20. Feshbach-Einstein Condensates

    SciTech Connect

    Rousseau, V. G.; Denteneer, P. J. H.

    2009-01-09

    We investigate the phase diagram of a two-species Bose-Hubbard model describing atoms and molecules on a lattice, interacting via a Feshbach resonance. We identify a region where the system exhibits an exotic super-Mott phase and regions with phases characterized by atomic and/or molecular condensates. Our approach is based on a recently developed exact quantum Monte Carlo algorithm: the stochastic Green function algorithm with tunable directionality. We confirm some of the results predicted by mean-field studies, but we also find disagreement with these studies. In particular, we find a phase with an atomic but no molecular condensate, which is missing in all mean-field phase diagrams.

  1. Multilayer graphene condenser microphone

    NASA Astrophysics Data System (ADS)

    Todorović, Dejan; Matković, Aleksandar; Milićević, Marijana; Jovanović, Djordje; Gajić, Radoš; Salom, Iva; Spasenović, Marko

    2015-12-01

    Vibrating membranes are the cornerstone of acoustic technology, forming the backbone of modern loudspeakers and microphones. Acoustic performance of a condenser microphone is derived mainly from the membrane’s size, surface mass and achievable static tension. The widely studied and available nickel has been a dominant membrane material for professional microphones for several decades. In this paper we introduce multilayer graphene as a membrane material for condenser microphones. The graphene device outperforms a high end commercial nickel-based microphone over a significant part of the audio spectrum, with a larger than 10 dB enhancement of sensitivity. Our experimental results are supported with numerical simulations, which also show that a 300 layer thick graphene membrane under maximum tension would offer excellent extension of the frequency range, up to 1 MHz.

  2. Condensation transition and forced unravelling of DNA-histone H1 toroids: a multi-state free energy landscape

    NASA Astrophysics Data System (ADS)

    Mack, A. H.; Schlingman, D. J.; Salinas, R. D.; Regan, L.; Mochrie, S. G. J.

    2015-02-01

    DNA is known to condense with multivalent cations and positively charged proteins. However, the properties and energetics of DNA superstructures, such as chromatin, are poorly understood. As a model system, we investigate histone H1 condensation of DNA with tethered particle motion and force-extension measurements. We show that after the addition of H1 to DNA, a concentration dependent lag time is followed by the DNA spontaneously condensing. The trigger for this condensation phase transition can be modeled as sufficient H1s having bound to the DNA, providing insight into the 30 nm fiber condensation upon H1 binding. Furthermore, optical tweezers force-extension measurements of histone H1 condensed DNA reveals a sequence of state transitions corresponding to the unwinding of superhelical turns. We determine the complete, experimental, multi-state free energy landscape for the complex using Crooks fluctuation theorem. The measured force-versus-extension and free energy landscape are compared to predictions from a simple, theoretical model. This work encourages the theoretical description of DNA/protein structure and energetics and their role in chromatin and other, more complex, systems.

  3. Chondrules as condensation products

    NASA Technical Reports Server (NTRS)

    Wood, J. A.; Mcsween, H. Y., Jr.

    1977-01-01

    The formation of meteoritic chondrules via condensation from the primordial solar nebula is discussed. Chondrule formation in regions where the gas/dust ratio was enhanced, and where transient high energy events heated the gas and temporarily vaporized the dust, is advocated. The observed diversity of chondrule types can be understood as resulting from local variations in the initial gas/dust proportions and other parameters.

  4. Bose-Einstein Condensation

    SciTech Connect

    El-Sherbini, Th.M.

    2005-03-17

    This article gives a brief review of Bose-Einstein condensation. It is an exotic quantum phenomenon that was observed in dilute atomic gases for the first time in 1995. It exhibits a new state of matter in which a group of atoms behaves as a single particle. Experiments on this form of matter are relevant to many different areas of physics- from atomic clocks and quantum computing to super fluidity, superconductivity and quantum phase transition.

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

    PubMed Central

    Zhou, Qi; Bachtrog, Doris

    2015-01-01

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

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

    PubMed

    Zhou, Qi; Bachtrog, Doris

    2015-06-01

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

  7. Asymmetric condensed dark matter

    NASA Astrophysics Data System (ADS)

    Aguirre, Anthony; Diez-Tejedor, Alberto

    2016-04-01

    We explore the viability of a boson dark matter candidate with an asymmetry between the number densities of particles and antiparticles. A simple thermal field theory analysis confirms that, under certain general conditions, this component would develop a Bose-Einstein condensate in the early universe that, for appropriate model parameters, could survive the ensuing cosmological evolution until now. The condensation of a dark matter component in equilibrium with the thermal plasma is a relativistic process, hence the amount of matter dictated by the charge asymmetry is complemented by a hot relic density frozen out at the time of decoupling. Contrary to the case of ordinary WIMPs, dark matter particles in a condensate must be lighter than a few tens of eV so that the density from thermal relics is not too large. Big-Bang nucleosynthesis constrains the temperature of decoupling to the scale of the QCD phase transition or above. This requires large dark matter-to-photon ratios and very weak interactions with standard model particles.

  8. Polymer Physics of the Large-Scale Structure of Chromatin.

    PubMed

    Bianco, Simona; Chiariello, Andrea Maria; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2016-01-01

    We summarize the picture emerging from recently proposed models of polymer physics describing the general features of chromatin large scale spatial architecture, as revealed by microscopy and Hi-C experiments. PMID:27659986

  9. Control of chromatin structure by long noncoding RNA

    PubMed Central

    Böhmdorfer, Gudrun; Wierzbicki, Andrzej T.

    2015-01-01

    Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via DNA methylation and posttranslational histone modifications is a well-documented function of lncRNA in plants, fungi, and animals. Here, we summarize evidence showing that lncRNA also controls chromatin structure including nucleosome positioning and chromosome looping. We focus on data from plant experimental systems, discussed in the context of other eukaryotes. We explain the mechanisms of lncRNA-controlled chromatin remodeling and the implications of the functional interplay between noncoding transcription and several different chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin modifications and structure. PMID:26410408

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

    PubMed

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

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations. PMID:22825423

  11. Developmental regulation of chromatin conformation by Hox proteins in Drosophila

    PubMed Central

    Agelopoulos, Marios; McKay, Daniel J.; Mann, Richard S.

    2012-01-01

    Summary We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunopreciptiate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5’ regulatory region is in close proximity to the Dll promoter. In Dll nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and non-expressing cell types, respectively. PMID:22523743

  12. In vitro binding of nitracrine to DNA in chromatin.

    PubMed

    Wilmańska, D; Szmigiero, L; Gniazdowski, M

    1989-01-01

    In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions. PMID:2742691

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

    PubMed

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

    2012-07-01

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

  14. The chromatin landscape of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Toth, Zsolt; Brulois, Kevin; Jung, Jae U

    2013-05-01

    Kaposi's sarcoma-associated herpesvirus is an oncogenic γ-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis. PMID:23698402

  15. Insights into Chromatin Structure and Dynamics in Plants

    PubMed Central

    Rosa, Stefanie; Shaw, Peter

    2013-01-01

    The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

  16. Polymer Physics of the Large-Scale Structure of Chromatin.

    PubMed

    Bianco, Simona; Chiariello, Andrea Maria; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2016-01-01

    We summarize the picture emerging from recently proposed models of polymer physics describing the general features of chromatin large scale spatial architecture, as revealed by microscopy and Hi-C experiments.

  17. ATP-dependent chromatin remodeling shapes the DNA replication landscape

    PubMed Central

    Vincent, Jack A.; Kwong, Tracey J.; Tsukiyama, Toshio

    2009-01-01

    Summary The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 play especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes promoting DNA replication, and define a specific stage of replication that requires remodeling for normal function. PMID:18408730

  18. Chromatin mechanisms in the developmental control of imprinted gene expression.

    PubMed

    Sanli, Ildem; Feil, Robert

    2015-10-01

    Hundreds of protein-coding genes and regulatory non-coding RNAs (ncRNAs) are subject to genomic imprinting. The mono-allelic DNA methylation marks that control imprinted gene expression are somatically maintained throughout development, and this process is linked to specific chromatin features. Yet, at many imprinted genes, the mono-allelic expression is lineage or tissue-specific. Recent studies provide mechanistic insights into the developmentally-restricted action of the 'imprinting control regions' (ICRs). At several imprinted domains, the ICR expresses a long ncRNA that mediates chromatin repression in cis (and probably in trans as well). ICRs at other imprinted domains mediate higher-order chromatin structuration that enhances, or prevents, transcription of close-by genes. Here, we present how chromatin and ncRNAs contribute to developmental control of imprinted gene expression and discuss implications for disease. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

  19. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  20. The Regulation of Chromatin by Dynamic SUMO Modifications.

    PubMed

    Wilson, Nicole R; Hochstrasser, Mark

    2016-01-01

    Protein modification by the small ubiquitin-related modifier (SUMO) protein regulates numerous cellular pathways and mounting evidence reveals a critical role for SUMO in modulating gene expression. Dynamic sumoylation of transcription factors, chromatin-modifying enzymes, histones, and other chromatin-associated factors significantly affects the transcriptional status of the eukaryotic genome. Recent studies have employed high-throughput ChIP-Seq analyses to gain clues regarding the role of the SUMO pathway in regulating chromatin-based transactions. Indeed, the global distribution of SUMO across chromatin reveals an important function for SUMO in controlling transcription, particularly of genes involved in protein synthesis. These newly appreciated patterns of genome-wide sumoylation will inform more directed studies aimed at analyzing how the dynamics of gene expression are controlled by posttranslational SUMO modification. PMID:27631795

  1. ISWI chromatin remodeling complexes in the DNA damage response.

    PubMed

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA.

  2. A Rare Stapes Abnormality

    PubMed Central

    Kanona, Hala; Virk, Jagdeep Singh; Kumar, Gaurav; Chawda, Sanjiv; Khalil, Sherif

    2015-01-01

    The aim of this study is to increase awareness of rare presentations, diagnostic difficulties alongside management of conductive hearing loss and ossicular abnormalities. We report the case of a 13-year-old female reporting progressive left-sided hearing loss and high resolution computed tomography was initially reported as normal. Exploratory tympanotomy revealed an absent stapedius tendon and lack of connection between the stapes superstructure and footplate. The footplate was fixed. Stapedotomy and stapes prosthesis insertion resulted in closure of the air-bone gap by 50 dB. A review of world literature was performed using MedLine. Middle ear ossicular discontinuity can result in significant conductive hearing loss. This can be managed effectively with surgery to help restore hearing. However, some patients may not be suitable or decline surgical intervention and can be managed safely conservatively. PMID:25628909

  3. CTCF depletion alters chromatin structure and transcription of myeloid-specific factors.

    PubMed

    Ouboussad, Lylia; Kreuz, Sarah; Lefevre, Pascal F

    2013-10-01

    Differentiation is a multistep process tightly regulated and controlled by complex transcription factor networks. Here, we show that the rate of differentiation of common myeloid precursor cells increases after depletion of CTCF, a protein emerging as a potential key factor regulating higher-order chromatin structure. We identified CTCF binding in the vicinity of important transcription factors regulating myeloid differentiation and showed that CTCF depletion impacts on the expression of these genes in concordance with the observed acceleration of the myeloid commitment. Furthermore, we observed a loss of the histone variant H2A.Z within the selected promoter regions and an increase in non-coding RNA transcription upstream of these genes. Both abnormalities suggest a global chromatin structure destabilization and an associated increase of non-productive transcription in response to CTCF depletion but do not drive the CTCF-mediated transcription alterations of the neighbouring genes. Finally, we detected a transient eviction of CTCF at the Egr1 locus in correlation with Egr1 peak of expression in response to lipopolysaccharide (LPS) treatment in macrophages. This eviction is also correlated with the expression of an antisense non-coding RNA transcribing through the CTCF-binding region indicating that non-coding RNA transcription could be the cause and the consequence of CTCF eviction.

  4. Nuclear Localization of PRDM9 and Its Role in Meiotic Chromatin Modifications and Homologous Synapsis

    PubMed Central

    Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G.; Hu, Jianjun; Saxl, Ruth L.; Baker, Christopher L.; Petkov, Petko M.; Paigen, Kenneth; Handel, Mary Ann

    2015-01-01

    Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc-finger domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ-cell nuclei at pre-leptonema to early leptonema, but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function, and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots. PMID:25894966

  5. Chromatin-Remodelling Complex NURF Is Essential for Differentiation of Adult Melanocyte Stem Cells.

    PubMed

    Koludrovic, Dana; Laurette, Patrick; Strub, Thomas; Keime, Céline; Le Coz, Madeleine; Coassolo, Sebastien; Mengus, Gabrielle; Larue, Lionel; Davidson, Irwin

    2015-10-01

    MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes. PMID:26440048

  6. Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

    PubMed

    He, Shuying; Limi, Saima; McGreal, Rebecca S; Xie, Qing; Brennan, Lisa A; Kantorow, Wanda Lee; Kokavec, Juraj; Majumdar, Romit; Hou, Harry; Edelmann, Winfried; Liu, Wei; Ashery-Padan, Ruth; Zavadil, Jiri; Kantorow, Marc; Skoultchi, Arthur I; Stopka, Tomas; Cvekl, Ales

    2016-06-01

    Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIβ, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation. PMID:27246713

  7. The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

    PubMed Central

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

  8. Chromatin-Remodelling Complex NURF Is Essential for Differentiation of Adult Melanocyte Stem Cells

    PubMed Central

    Koludrovic, Dana; Laurette, Patrick; Strub, Thomas; Keime, Céline; Le Coz, Madeleine; Coassolo, Sebastien; Mengus, Gabrielle; Larue, Lionel; Davidson, Irwin

    2015-01-01

    MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes. PMID:26440048

  9. The chromatin regulator Brpf1 regulates embryo development and cell proliferation.

    PubMed

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-05-01

    With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.

  10. Chromatin-Remodelling Complex NURF Is Essential for Differentiation of Adult Melanocyte Stem Cells.

    PubMed

    Koludrovic, Dana; Laurette, Patrick; Strub, Thomas; Keime, Céline; Le Coz, Madeleine; Coassolo, Sebastien; Mengus, Gabrielle; Larue, Lionel; Davidson, Irwin

    2015-10-01

    MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes.

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

    PubMed Central

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

    2014-01-01

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

  12. Modeling co-occupancy of transcription factors using chromatin features

    PubMed Central

    Liu, Liang; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

    Regulation of gene expression requires both transcription factor (TFs) and epigenetic modifications, and interplays between the two types of factors have been discovered. However study of relationships between chromatin features and TF–TF co-occupancy remains limited. Here, we revealed the relationship by first illustrating distinct profile patterns of chromatin features related to different binding events, including single TF binding and TF–TF co-occupancy of 71 TFs from five human cell lines. We further implemented statistical analyses to demonstrate the relationship by accurately predicting co-occupancy genome-widely using chromatin features including DNase I hypersensitivity, 11 histone modifications (HMs) and GC content. Remarkably, our results showed that the combination of chromatin features enables accurate predictions across the five cells. For individual chromatin features, DNase I enables high and consistent predictions. H3K27ac, H3K4me 2, H3K4me3 and H3K9ac are more reliable predictors than other HMs. Although the combination of 11 HMs achieves accurate predictions, their predictive ability varies considerably when a model obtained from one cell is applied to others, indicating relationship between HMs and TF–TF co-occupancy is cell type dependent. GC content is not a reliable predictor, but the addition of GC content to any other features enhances their predictive ability. Together, our results elucidate a strong relationship between TF–TF co-occupancy and chromatin features. PMID:26590261

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

    PubMed Central

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

    2016-01-01

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

  14. Epigenetic regulation of open chromatin in pluripotent stem cells.

    PubMed

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2015-01-01

    The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, alter, eventually, 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 open globally 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, including 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 genomewide nucleosome accessibility and nucleosome positioning. Greater understanding of epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem cells.

  15. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

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

    PubMed

    Zeng, Zixian; Jiang, Jiming

    2016-06-01

    Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum vulgare) by using a 2'-deoxy-2'fluoro-ribonucleotides (2'-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barley centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alpha-cenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed. PMID:27142171

  17. Compact structure of ribosomal chromatin in Xenopus laevis.

    PubMed Central

    Spadafora, C; Crippa, M

    1984-01-01

    Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation. Images PMID:6709502

  18. Environmental-stress-induced Chromatin Regulation and its Heritability

    PubMed Central

    Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

    2014-01-01

    Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives. PMID:25045581

  19. Environmental-stress-induced Chromatin Regulation and its Heritability.

    PubMed

    Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

    2014-01-15

    Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives.

  20. Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

    PubMed Central

    Nguyen, Uyen T. T.; Bittova, Lenka; Müller, Manuel M.; Fierz, Beat; David, Yael; Houck-Loomis, Brian; Feng, Vanessa; Dann, Geoffrey P.; Muir, Tom W.

    2014-01-01

    Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin. PMID:24997861

  1. Antagonistic roles of Drosophila Tctp and Brahma in chromatin remodelling and stabilizing repeated sequences

    PubMed Central

    Hong, Sung-Tae; Choi, Kwang-Wook

    2016-01-01

    Genome stability is essential for all organisms. Translationally controlled tumour protein (TCTP) is a conserved protein associated with cancers. TCTP is involved in multiple intracellular functions, but its role in transcription and genome stability is poorly understood. Here, we demonstrate new functions of Drosophila TCTP (Tctp) in transcription and the stability of repeated sequences (rDNA and pericentromeric heterochromatin). Tctp binds Brahma (Brm) chromatin remodeler to negatively modulate its activity. Tctp mutants show abnormally high levels of transcription in a large set of genes and transposons. These defects are ameliorated by brm mutations. Furthermore, Tctp promotes the stability of repeated sequences by opposing the Brm function. Additional regulation of pericentromeric heterochromatin by Tctp is mediated by su(var)3-9 transcriptional regulation. Altogether, Tctp regulates transcription and the stability of repeated sequences by antagonizing excess Brm activity. This study provides insights into broader nuclear TCTP functions for the maintenance of genome stability. PMID:27687497

  2. Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

    PubMed

    Guerrero, Georgina; Delgado-Olguín, Paul; Escamilla-Del-Arenal, Martín; Furlan-Magaril, Mayra; Rebollar, Eria; De La Rosa-Velázquez, Inti A; Soto-Reyes, Ernesto; Rincón-Arano, Héctor; Valdes-Quezada, Christian; Valadez-Graham, Viviana; Recillas-Targa, Félix

    2007-07-01

    At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.

  3. The effect of heracleum persicum (Golpar) oil and alcoholic extracts on sperm parameters and chromatin quality in mice

    PubMed Central

    Taghizabet, Neda; Mangoli, Esmat; Anbari, Fatemeh; Masoodi, Seyed Ali; Talebi, Ali Reza; Mazrooei, Malihe

    2016-01-01

    Background: Evaluating the significance and the effects of plant-derived drugs on laboratory animal’s fertility was recognized. There was antioxidant activity reported from Heracleum persicum (Golpar). Objective: Current study aims to study the antioxidant effect of Golpar extracts on sperm parameters and chromatin quality in mice. Materials and Methods: Eighteen adult male mice were divided to 3 groups (10 wk old, 35 gr weight): group1 received hydro alcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 mg/kg, ip) and group 3 serving as the sham control group that received sterile water. Finally, left cauda epididymis of each animal was dissected and sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3) and acridine orange (AO) staining. Results: Progressive and non-progressive sperm motility were significantly increased in group 1 in comparison with group 3 (p=0.032). There was an increasing trend in progressive sperm motility and decreasing trend in non-progressive sperm motility in group 2 in comparison with group 3, but the differences were not significant (p=0.221 and p=0.144, respectively). According to the sperm chromatin quality, the results of TB and AO tests revealed significant differences (p=0.004, p=0.000, respectively) between those groups and showed that the extracts of Golpar cause DNA damage, but no differences can be observed between them in AB and CMA3 staining (p>0.05). Conclusion: The results showed that Heracleum persicum extracts may improve sperm motility. Also, it has harmful effects on sperm chromatin condensation and DNA integrity in mice. PMID:27525319

  4. Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.

    PubMed

    Fu, Hongxia; Freedman, Benjamin S; Lim, Chwee Teck; Heald, Rebecca; Yan, Jie

    2011-06-01

    Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures. PMID:21369955

  5. DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

    PubMed

    Mandel, R; Kolomijtseva, G; Brahms, J G

    1979-05-15

    Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.

  6. Effect of spontaneous condensation on condensation heat transfer in the presence of non-condensable gases

    SciTech Connect

    Karl, J.; Hein, D.

    1999-07-01

    The presence of non condensable gases like nitrogen or air reduces the condensation heat transfer during condensation of binary steam mixtures. The non condensable gas accumulates in the vapor phase boundary layer and causes a high heat transfer resistance. Especially with high pressures and low water temperatures spontaneous condensation reduces heat transfer additionally. Fog forms within the steam-nitrogen boundary layer and the steam condenses on the water droplets of the fog layer. The convective mass transfer to the cooling water interface diminishes. Raman spectroscopy and film theory are used to quantify this effect locally. The calculation of overall condensation rates in large steam nitrogen systems requires to use three dimensional CFD codes. The paper presents equations to predict fog formation in the boundary layer which can be implemented in CFD codes.

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  8. Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates

    PubMed Central

    Morrell, Jane M; Johannisson, Anders; Dalin, Anne-Marie; Hammar, Linda; Sandebert, Thomas; Rodriguez-Martinez, Heriberto

    2008-01-01

    Background Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to investigate the possibility of using these tests predictively. Methods Aliquots from fortyone ejaculates, obtained as part of the normal semen collection schedule at the Swedish National Stud, were used for morphological analysis by light microscopy, whereas thirtyseven were used for chromatin analysis (SCSA) by flow cytometry. The outcome of inseminations using these ejaculates was made available later in the same year. Results Ranges for the different parameters were as follows; normal morphology, 27–79.5%; DNA-fragmentation index (DFI), 4.8–19.0%; standard deviation of DNA fragmentation index (SD_DFI) 41.5–98.9, and mean of DNA fragmentation index (mean_DFI), 267.7–319.5. There was considerable variation among stallions, which was statistically significant for all these parameters except for mean_DFI (P < 0.001, P < 0.01, P < 0.001 and P < 0.2 respectively). There was a negative relationship between normal morphology and DFI (P < 0.05), between normal morphology and SD_DFI (P < 0.001), and between normal morphology and mean_DFI (P < 0.05). For specific defects, there was a direct relationship between the incidence of pear-shaped sperm heads and DFI (P < 0.05), and also nuclear pouches and DFI (P < 0.001), indicating that either morphological analysis or chromatin analysis was able to identify abnormalities in spermiogenesis that could compromise DNA

  9. Ictal Cardiac Ryhthym Abnormalities

    PubMed Central

    Ali, Rushna

    2016-01-01

    Cardiac rhythm abnormalities in the context of epilepsy are a well-known phenomenon. However, they are under-recognized and often missed. The pathophysiology of these events is unclear. Bradycardia and asystole are preceded by seizure onset suggesting ictal propagation into the cortex impacting cardiac autonomic function, and the insula and amygdala being possible culprits. Sudden unexpected death in epilepsy (SUDEP) refers to the unanticipated death of a patient with epilepsy not related to status epilepticus, trauma, drowning, or suicide. Frequent refractory generalized tonic-clonic seizures, anti-epileptic polytherapy, and prolonged duration of epilepsy are some of the commonly identified risk factors for SUDEP. However, the most consistent risk factor out of these is an increased frequency of generalized tonic–clonic seizures (GTC). Prevention of SUDEP is extremely important in patients with chronic, generalized epilepsy. Since increased frequency of GTCS is the most consistently reported risk factor for SUDEP, effective seizure control is the most important preventive strategy. PMID:27347227

  10. Ictal Cardiac Ryhthym Abnormalities.

    PubMed

    Ali, Rushna

    2016-01-01

    Cardiac rhythm abnormalities in the context of epilepsy are a well-known phenomenon. However, they are under-recognized and often missed. The pathophysiology of these events is unclear. Bradycardia and asystole are preceded by seizure onset suggesting ictal propagation into the cortex impacting cardiac autonomic function, and the insula and amygdala being possible culprits. Sudden unexpected death in epilepsy (SUDEP) refers to the unanticipated death of a patient with epilepsy not related to status epilepticus, trauma, drowning, or suicide. Frequent refractory generalized tonic-clonic seizures, anti-epileptic polytherapy, and prolonged duration of epilepsy are some of the commonly identified risk factors for SUDEP. However, the most consistent risk factor out of these is an increased frequency of generalized tonic-clonic seizures (GTC). Prevention of SUDEP is extremely important in patients with chronic, generalized epilepsy. Since increased frequency of GTCS is the most consistently reported risk factor for SUDEP, effective seizure control is the most important preventive strategy. PMID:27347227

  11. Communication and abnormal behaviour.

    PubMed

    Crown, S

    1979-01-01

    In this paper the similarities between normal and abnormal behaviour are emphasized and selected aspects of communication, normal and aberrant, between persons are explored. Communication in a social system may be verbal or non-verbal: one person's actions cause a response in another person. This response may be cognitive, behavioural or physiological. Communication may be approached through the individual, the social situation or social interaction. Psychoanalysis approaches the individual in terms of the coded communications of psychoneurotic symptoms or psychotic behaviour; the humanist-existential approach is concerned more with emotional expression. Both approaches emphasize the development of individual identity. The interaction between persons and their social background is stressed. Relevant are sociological concepts such as illness behaviour, stigma, labelling, institutionalization and compliance. Two approaches to social interactions are considered: the gamesplaying metaphor, e.g. back pain as a psychosocial manipulation--the 'pain game'; and the 'spiral of reciprocal perspectives' which emphasizes the interactional complexities of social perceptions. Communicatory aspects of psychological treatments are noted: learning a particular metaphor such as 'resolution' of the problem (psychotherapy), learning more 'rewarding' behaviour (learning theory) or learning authenticity or self-actualization (humanist-existential).

  12. Communication and abnormal behaviour.

    PubMed

    Crown, S

    1979-01-01

    In this paper the similarities between normal and abnormal behaviour are emphasized and selected aspects of communication, normal and aberrant, between persons are explored. Communication in a social system may be verbal or non-verbal: one person's actions cause a response in another person. This response may be cognitive, behavioural or physiological. Communication may be approached through the individual, the social situation or social interaction. Psychoanalysis approaches the individual in terms of the coded communications of psychoneurotic symptoms or psychotic behaviour; the humanist-existential approach is concerned more with emotional expression. Both approaches emphasize the development of individual identity. The interaction between persons and their social background is stressed. Relevant are sociological concepts such as illness behaviour, stigma, labelling, institutionalization and compliance. Two approaches to social interactions are considered: the gamesplaying metaphor, e.g. back pain as a psychosocial manipulation--the 'pain game'; and the 'spiral of reciprocal perspectives' which emphasizes the interactional complexities of social perceptions. Communicatory aspects of psychological treatments are noted: learning a particular metaphor such as 'resolution' of the problem (psychotherapy), learning more 'rewarding' behaviour (learning theory) or learning authenticity or self-actualization (humanist-existential). PMID:261653

  13. Abnormal uterine bleeding.

    PubMed

    Whitaker, Lucy; Critchley, Hilary O D

    2016-07-01

    Abnormal uterine bleeding (AUB) is a common and debilitating condition with high direct and indirect costs. AUB frequently co-exists with fibroids, but the relationship between the two remains incompletely understood and in many women the identification of fibroids may be incidental to a menstrual bleeding complaint. A structured approach for establishing the cause using the Fédération International de Gynécologie et d'Obstétrique (FIGO) PALM-COEIN (Polyp, Adenomyosis, Leiomyoma, Malignancy (and hyperplasia), Coagulopathy, Ovulatory disorders, Endometrial, Iatrogenic and Not otherwise classified) classification system will facilitate accurate diagnosis and inform treatment options. Office hysteroscopy and increasing sophisticated imaging will assist provision of robust evidence for the underlying cause. Increased availability of medical options has expanded the choice for women and many will no longer need to recourse to potentially complicated surgery. Treatment must remain individualised and encompass the impact of pressure symptoms, desire for retention of fertility and contraceptive needs, as well as address the management of AUB in order to achieve improved quality of life. PMID:26803558

  14. Abortion for fetal abnormality.

    PubMed

    Maclean, N E

    1979-07-25

    I wish to thank Dr. Pauline Bennett for her reply (NZ Med J, 13 June). She has demonstrated well that in dealing with sensitive difficult issues such as abortion for fetal abnormality, the one thing the doctor is not recommended to do is to speak the truth] I am prompted to write this letter for 2 reasons. Firstly, the excellent letter written by Dr. A. M. Rutherford (NZ Med J, 13 June) on the subject of abortion stated, "The most disturbing feature about the whole controversy is the 'blunting of our conscience'." When the doctors are not encouraged to be honest with patients then indeed our conscience has been blunted. Secondly, I watched Holocaust last night, and cannot refrain from stating that I see frightening parallels between our liberal abortion policy and the activities of the Nazis. As I watched the "mental patients" being herded into the shed for gassing by the polite, tidy, white coated medical staff, and then heard the compassionate, sensitive, letter of the hospital authorities to the relatives of the deceased, the parallel became obvious. The mental patients were weak, defenseless, burdensome, and uneconomic; the unborn are weak, defenseless, burdensome, and uneconomic. The hospital authority's letter was acceptable in many ways, acceptable except that its words bore no relation to the truth. It is said that the "first casualty of war is the truth". Whether that war involves the Jews, or the insane, or the unborn, the statement would seem correct.

  15. Condensed Matter Nuclear Science

    NASA Astrophysics Data System (ADS)

    Biberian, Jean-Paul

    2006-02-01

    1. General. A tribute to gene Mallove - the "Genie" reactor / K. Wallace and R. Stringham. An update of LENR for ICCF-11 (short course, 10/31/04) / E. Storms. New physical effects in metal deuterides / P. L. Hagelstein ... [et al.]. Reproducibility, controllability, and optimization of LENR experiments / D. J. Nagel -- 2. Experiments. Electrochemistry. Evidence of electromagnetic radiation from Ni-H systems / S. Focardi ... [et al.]. Superwave reality / I. Dardik. Excess heat in electrolysis experiments at energetics technologies / I. Dardik ... [et al.]. "Excess heat" during electrolysis in platinum/K[symbol]CO[symbol]/nickel light water system / J. Tian ... [et al.]. Innovative procedure for the, in situ, measurement of the resistive thermal coefficient of H(D)/Pd during electrolysis; cross-comparison of new elements detected in the Th-Hg-Pd-D(H) electrolytic cells / F. Celani ... [et al.]. Emergence of a high-temperature superconductivity in hydrogen cycled Pd compounds as an evidence for superstoihiometric H/D sites / A. Lipson ... [et al.]. Plasma electrolysis. Calorimetry of energy-efficient glow discharge - apparatus design and calibration / T. B. Benson and T. O. Passell. Generation of heat and products during plasma electrolysis / T. Mizuno ... [et al.]. Glow discharge. Excess heat production in Pd/D during periodic pulse discharge current in various conditions / A. B. Karabut. Beam experiments. Accelerator experiments and theoretical models for the electron screening effect in metallic environments / A. Huke, K. Czerski, and P. Heide. Evidence for a target-material dependence of the neutron-proton branching ratio in d+d reactions for deuteron energies below 20keV / A. Huke ... [et al.]. Experiments on condensed matter nuclear events in Kobe University / T. Minari ... [et al.]. Electron screening constraints for the cold fusion / K. Czerski, P. Heide, and A. Huke. Cavitation. Low mass 1.6 MHz sonofusion reactor / R. Stringham. Particle detection. Research

  16. The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

    PubMed Central

    Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

    2016-01-01

    Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

  17. Rejoining and misrejoining of radiation-induced chromatin breaks. IV. Charged particles

    NASA Technical Reports Server (NTRS)

    Durante, M.; Furusawa, Y.; George, K.; Gialanella, G.; Greco, O.; Grossi, G.; Matsufuji, N.; Pugliese, M.; Yang, T. C.

    1998-01-01

    We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

  18. Confinement Contains Condensates

    SciTech Connect

    Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.

    2012-03-12

    Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

  19. Expansion in condensates

    SciTech Connect

    Chakrabarti, J.; Sajjad Zahir, M.

    1985-03-01

    We show that the product of local current operators in quantum chromodynamics (QCD), when expanded in terms of condensates, such as psi-barpsi, G/sup a//sub munu/ G/sup a//sub munu/, psi-barGAMMA psipsi-barGAMMApsi, f/sub a/bcG/sup a//sub munu/G/sup b//sub nualpha/ x G/sup c//sub alphamu/, etc., yields a series in Planck's constant. This, however, provides no hint that the higher terms in such an expansion may be less significant.

  20. Condensed Plasmas under Microgravity

    NASA Technical Reports Server (NTRS)

    Morfill, G. E.; Thomas, H. M.; Konopka, U.; Rothermel, H.; Zuzic, M.; Ivlev, A.; Goree, J.; Rogers, Rick (Technical Monitor)

    1999-01-01

    Experiments under microgravity conditions were carried out to study 'condensed' (liquid and crystalline) states of a colloidal plasma (ions, electrons, and charged microspheres). Systems with approximately 10(exp 6) microspheres were produced. The observed systems represent new forms of matter--quasineutral, self-organized plasmas--the properties of which are largely unexplored. In contrast to laboratory measurements, the systems under microgravity are clearly three dimensional (as expected); they exhibit stable vortex flows, sometimes adjacent to crystalline regions, and a central 'void,' free of microspheres.

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

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

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

  2. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    PubMed

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional

  3. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity

    PubMed Central

    Kim, Tae-Hee; Li, Fugen; Ferreiro-Neira, Isabel; Ho, Li-Lun; Luyten, Annouck; Nalapareddy, Kodandaramireddy; Long, Henry; Verzi, Michael; Shivdasani, Ramesh A.

    2014-01-01

    Cells differentiate when transcription factors (TFs) bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem (ES) cells, chromatin at lineage-restricted genes becomes sequentially accessible1-4, probably by virtue of “pioneer” TF activity5, but tissues may utilize other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbors6, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where Notch signaling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages7-9. Transcript profiles in isolated LGR5+ intestinal stem cells (ISC)10 and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNaseI hypersensitivity - signifying accessible, permissive chromatin - at most of the same cis-elements. Enhancers acting uniquely in progenitors were well-demarcated in LGR5+ ISC, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific TF, controls lateral inhibition through Delta-like Notch ligand genes and also drives numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to presence or absence of a key TF. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted TF with broadly permissive chromatin established

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

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

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

  5. Quantitative assessment of DNA condensation.

    PubMed

    Trubetskoy, V S; Slattum, P M; Hagstrom, J E; Wolff, J A; Budker, V G

    1999-02-15

    A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.

  6. Mitotic chromosome structure and condensation.

    PubMed

    Belmont, Andrew S

    2006-12-01

    Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization. PMID:17046228

  7. Strange Disoriented Chiral Condensates

    NASA Astrophysics Data System (ADS)

    Abdel-Aziz, Mohamed; Gavin, Sean

    2002-10-01

    Enhancement of omega and anti-omega baryon production in Pb+Pb collisions at the CERN SPS can be explained by the formation of many small regions of disordered chiral condensate. This explanation implies that neutral and charged kaons as well as pions must exhibit novel isospin fluctuations. Fluctuations due to transient behavior of the Polyakov Loop condensate can produce similar phenomena. Kapusta and Gavin have computed the distribution of the fraction of neutral pions and kaons from such regions. We proposed robust statistical observables that can be used to extract the novel isospin fluctuations from background contributions in neutral/charged pion and K-short/K-charged correlation measurements at RHIC and LHC. The STAR experiment is currently examining K-short/K-charged correlations. Note that Pruneau, Voloshin and Gavin have proposed similar observables to study net-charge fluctuations. To obtain a baseline for comparison to RHIC and SPS experiments, Abdel-Aziz and Gavin compute these observables using numerical simulations using HIJING and URQMD event generators. We also obtain limits on the size and number of disordered regions by comparing to photon and charged-pion searches from WA98 and other SPS experiments. We will compare to the first results from STAR K-short/K-charged analysis.

  8. Kaon Condensation with Lattice QCD

    SciTech Connect

    Detmold, Will; Detmold, William; Detmold, Will; Detmold, William; Savage, Martin; Walker-Loud, Andre; Orginos, Konstantinos; Torok, Aaron

    2008-09-01

    doi: http://dx.doi.org/10.1103/PhysRevD.78.054514
    Kaon condensation may play an important role in the structure of hadronic matter at densities greater than that of nuclear matter, as exist in the interior of neutron stars. We present the results of the first lattice QCD calculation of kaon condensation obtained by studying systems containing up to twelve charged kaons. Surprisingly, the equation of state of the condensate is remarkably well reproduced by leading order chiral perturbation theory. We determine the three-kaon interaction from the multi-kaon systems and update our results for pion condensates.

  9. Haem degradation in abnormal haemoglobins.

    PubMed Central

    Brown, S B; Docherty, J C

    1978-01-01

    The coupled oxidation of certain abnormal haemoglobins leads to different bile-pigment isomer distributions from that of normal haemoglobin. The isomer pattern may be correlated with the structure of the abnormal haemoglobin in the neighbourhood of the haem pocket. This is support for haem degradation by an intramolecular reaction. PMID:708385

  10. Systemic abnormalities in liver disease

    PubMed Central

    Minemura, Masami; Tajiri, Kazuto; Shimizu, Yukihiro

    2009-01-01

    Systemic abnormalities often occur in patients with liver disease. In particular, cardiopulmonary or renal diseases accompanied by advanced liver disease can be serious and may determine the quality of life and prognosis of patients. Therefore, both hepatologists and non-hepatologists should pay attention to such abnormalities in the management of patients with liver diseases. PMID:19554648

  11. Abnormal pressure in hydrocarbon environments

    USGS Publications Warehouse

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  12. Electrocardiograph abnormalities revealed during laparoscopy.

    PubMed

    Nijjer, Sukhjinder; Dubrey, Simon William

    2010-01-01

    This brief case presents a well patient in whom an electrocardiograph abnormality consistent with an accessory pathway was found during a routine procedure. We present the electrocardiographs, explain the underlying condition, and consider why the abnormality was revealed in this manner.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. Histone modifications and chromatin dynamics: a focus on filamentous fungi

    PubMed Central

    Brosch, Gerald; Loidl, Peter; Graessle, Stefan

    2008-01-01

    The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. PMID:18221488

  16. Role of chromatin in water stress responses in plants.

    PubMed

    Han, Soon-Ki; Wagner, Doris

    2014-06-01

    As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth.

  17. Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes.

    PubMed

    Lee, Kyungeun; Kim, Deok Ryong; Ahn, Byungchan

    2004-08-31

    The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities. PMID:15359130

  18. Changes in chromatin structure associated with Alzheimer's disease.

    PubMed

    Lewis, P N; Lukiw, W J; De Boni, U; McLachlan, D R

    1981-11-01

    The enzyme micrococcal nuclease was used to examine the accessibility of chromatin extracted from brains of 13 patients with senile and presenile dementia of the Alzheimer type. Compared with chromatin extracted from brains of 8 patients without neurological signs or brain pathology and brains of 7 patients with nonAlzheimer dementia, Alzheimer chromatin was less accessible to this enzyme. Reduced accessibility was reflected by a reduced yield of mononucleosomes in comparison with dinucleosomes and larger oligomers. Both neuronal and glial chromatin were found to be similarly affected. The reduced yield of mononucleosomes from Alzheimer chromatin is not due to their increased breakdown, but is probably related to protein associated with the internucleosomal linker region that retards nuclease action. Dinucleosomes isolated from control and Alzheimer nuclease digests were examined for their protein complement. Three perchloric acid-soluble proteins situated in the histone H1 region of sodium dodecyl sulfate (SDS) gels were present in elevated levels in Alzheimer dinucleosomes. These results represent the first example of altered chromosomal proteins associated with a diseased state of the brain.

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

    PubMed Central

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

    2015-01-01

    The chromatin fiber undergoes significant structural changes during the cell’s life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous. PMID:25564319

  20. Circadian rhythms and memory formation: regulation by chromatin remodeling.

    PubMed

    Sahar, Saurabh; Sassone-Corsi, Paolo

    2012-01-01

    Epigenetic changes, such as DNA methylation or histone modification, can remodel the chromatin and regulate gene expression. Remodeling of chromatin provides an efficient mechanism of transducing signals, such as light or nutrient availability, to regulate gene expression. CLOCK:BMAL1 mediated activation of clock-controlled genes (CCGs) is coupled to circadian changes in histone modification at their promoters. Several chromatin modifiers, such as the deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be recruited to the promoters of the CCGs in a circadian manner. Interestingly, the central element of the core clock machinery, the transcription factor CLOCK, also possesses histone acetyltransferase activity. Rhythmic expression of the CCGs is abolished in the absence of these chromatin modifiers. Recent research has demonstrated that chromatin remodeling is at the cross-roads of circadian rhythms and regulation of metabolism and aging. It would be of interest to identify if similar pathways exist in the epigenetic regulation of memory formation. PMID:22470318

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

    PubMed

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

    2012-12-13

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

  2. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  3. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  4. Condensation Processes in Geothermal Systems

    NASA Astrophysics Data System (ADS)

    Norman, D. I.; Moore, J. N.

    2005-12-01

    We model condensation processes in geothermal systems to understand how this process changes fluid chemistry. We assume two processes operate in geothermal systems: 1) condensation of a vapor phase derived by boiling an aqueous geothermal fluid into a cool near surface water and 2) condensation of a magmatic vapor by a deep circulating meteoric thermal fluid. It is assumed that the condensation process has two stages. Initially the condensing fluid is under saturated in gaseous species. Condensation of the vapor phase continues until the pressure on the fluid equals the sum of the partial pressures of water and the dissolved gaseous species. At that time bubbles flux through the condensing fluid. In time the fluid and fluxing gas phase come to equilibrium. Calculation shows that during the second stage of the condensation process the liquid phase becomes enriched in more soluble gaseous species like CO2 and H2S, and depleted in less soluble species like CH4 and N2. Stage 2 condensation processes can therefore be monitored by ratios of more and less condensable species like CO2/N2. Condensation of vapor released by boiling geothermal fluids results in liquids with high concentrations of H2S and CO2 like is seen in geothermal system steam-heated waters. Condensation of a magmatic vapor into circulating meteoric water has been proposed, but not well demonstrated. We compare to our models the Cerro Prieto, Mexico gas analysis data set collected over twelve years time by USGS personnel. It was assumed for modeling that the Cerro Prieto geothermal fluids are circulating meteoritic fluids with N2/Ar ratios about 40 to which is added a magmatic vapor with N2/Ar ratio = 400. The Cerro Prieto analyses show a strong correlation between N2/Ar and CO2/N2 as predicted by calculation. Two dimensional image plots of well N2/Ar + CO2/N2 show a bull's-eye pattern on the geothermal field. Image plots of analyses collected over a year or less time show N2/Ar and CO2/N2 hot spots

  5. Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry

    SciTech Connect

    Evenson, D.P.; Baer, R.K.; Jost, L.K. )

    1989-01-01

    The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, or 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated with dose and with each other. Sperm head morphology and sperm chromatic structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

  6. Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET).

    PubMed

    Choy, Jocelyn; Fullwood, Melissa J

    2017-01-01

    Genomic DNA is dynamically associated with protein factors and folded to form chromatin fibers. The 3-dimensional (3D) configuration of the chromatin will enable the distal genetic elements to come into close proximity, allowing transcriptional regulation. Noncoding RNA can mediate the 3D structure of chromatin. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a valuable and powerful technique in molecular biology which allows the study of unbiased, genome-wide de novo chromatin interactions with paired-end tags. Here, we describe the standard version of ChIA-PET and a Multiplex ChIA-PET version. PMID:27662871

  7. Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum, structure of chromatin, 3H-lysine and 3H-arginine incorporation.

    PubMed

    Kwiatkowska, Maria; Popłońska, Katarzyna

    2002-01-01

    On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from

  8. Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei*

    PubMed Central

    Stanne, Tara; Narayanan, Mani Shankar; Ridewood, Sophie; Ling, Alexandra; Witmer, Kathrin; Kushwaha, Manish; Wiesler, Simone; Wickstead, Bill; Wood, Jennifer; Rudenko, Gloria

    2015-01-01

    ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution. PMID:26378228

  9. Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei.

    PubMed

    Stanne, Tara M; Narayanan, Mani Shankar; Ridewood, Sophie; Ling, Alexandra; Witmer, Kathrin; Kushwaha, Manish; Wiesler, Simone; Wickstead, Bill; Wood, Jennifer; Rudenko, Gloria

    2015-11-01

    ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.

  10. Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei.

    PubMed

    Stanne, Tara M; Narayanan, Mani Shankar; Ridewood, Sophie; Ling, Alexandra; Witmer, Kathrin; Kushwaha, Manish; Wiesler, Simone; Wickstead, Bill; Wood, Jennifer; Rudenko, Gloria

    2015-11-01

    ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution. PMID:26378228

  11. Condenser for photolithography system

    SciTech Connect

    Sweatt, William C.

    2004-03-02

    A condenser for a photolithography system, in which a mask image from a mask is projected onto a wafer through a camera having an entrance pupil, includes a source of propagating radiation, a first mirror illuminated by the radiation, a mirror array illuminated by the radiation reflected from said first mirror, and a second mirror illuminated by the radiation reflected from the array. The mirror array includes a plurality of micromirrors. Each of the micromirrors is selectively actuatable independently of each other. The first mirror and the second mirror are disposed such that the source is imaged onto a plane of the mask and the mirror array is imaged into the entrance pupil of the camera.

  12. Microgravity condensing heat exchanger

    NASA Technical Reports Server (NTRS)

    Thomas, Christopher M. (Inventor); Ma, Yonghui (Inventor); North, Andrew (Inventor); Weislogel, Mark M. (Inventor)

    2011-01-01

    A heat exchanger having a plurality of heat exchanging aluminum fins with hydrophilic condensing surfaces which are stacked and clamped between two cold plates. The cold plates are aligned radially along a plane extending through the axis of a cylindrical duct and hold the stacked and clamped portions of the heat exchanging fins along the axis of the cylindrical duct. The fins extend outwardly from the clamped portions along approximately radial planes. The spacing between fins is symmetric about the cold plates, and are somewhat more closely spaced as the angle they make with the cold plates approaches 90.degree.. Passageways extend through the fins between vertex spaces which provide capillary storage and communicate with passageways formed in the stacked and clamped portions of the fins, which communicate with water drains connected to a pump externally to the duct. Water with no entrained air is drawn from the capillary spaces.

  13. TALE proteins bind to both active and inactive chromatin.

    PubMed

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  14. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Chromatin and DNA sequences in defining promoters for transcription initiation.

    PubMed

    Müller, Ferenc; Tora, Làszlò

    2014-03-01

    One of the key events in eukaryotic gene regulation and consequent transcription is the assembly of general transcription factors and RNA polymerase II into a functional pre-initiation complex at core promoters. An emerging view of complexity arising from a variety of promoter associated DNA motifs, their binding factors and recent discoveries in characterising promoter associated chromatin properties brings an old question back into the limelight: how is a promoter defined? In addition to position-dependent DNA sequence motifs, accumulating evidence suggests that several parallel acting mechanisms are involved in orchestrating a pattern marked by the state of chromatin and general transcription factor binding in preparation for defining transcription start sites. In this review we attempt to summarise these promoter features and discuss the available evidence pointing at their interactions in defining transcription initiation in developmental contexts. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

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

    PubMed

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

    2016-08-01

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. PMID:27445307

  20. Higher-order structure of Saccharomyces cerevisiae chromatin

    SciTech Connect

    Lowary, P.T.; Widom, J. )

    1989-11-01

    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.

  1. Human pescadillo induces large-scale chromatin unfolding.

    PubMed

    Zhang, Hao; Fang, Yan; Huang, Cuifen; Yang, Xiao; Ye, Qinong

    2005-06-01

    The human pescadillo gene encodes a protein with a BRCT domain. Pescadillo plays an important role in DNA synthesis, cell proliferation and transformation. Since BRCT domains have been shown to induce chromatin large-scale unfolding, we tested the role of Pescadillo in regulation of large-scale chromatin unfolding. To this end, we isolated the coding region of Pescadillo from human mammary MCF10A cells. Compared with the reported sequence, the isolated Pescadillo contains in-frame deletion from amino acid 580 to 582. Targeting the Pescadillo to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity maps to the BRCT domain of Pescadillo. These data provide a new clue to understanding the vital role of Pescadillo.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-08-01

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

  4. Amine catalyzed condensation of tetraethylorthosilicate

    NASA Technical Reports Server (NTRS)

    Jones, S.

    2001-01-01

    The catalysis of the condensation of hydrolyzed metal alkoxides by amines has been mentioned in the literature, but there has been no systematic study of their influence on the rate of the condensation reaction of the alkoxide and the microstructure of the resultant gel.

  5. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

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

    SciTech Connect

    Smith, P.J.

    1986-03-01

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

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

    PubMed

    Levi, Valeria; Gratton, Enrico

    2008-01-01

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

  8. Chromatin dynamics during interphase explored by single particle tracking

    PubMed Central

    Levi, Valeria; Gratton, Enrico

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2000-12-01

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

  10. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

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

  11. Rapid genome-scale mapping of chromatin accessibility in tissue

    PubMed Central

    2012-01-01

    Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of

  12. APPARATUS FOR CONDENSATION AND SUBLIMATION

    DOEpatents

    Schmidt, R.J.; Fuis, F. Jr.

    1958-10-01

    An apparatus is presented for the sublimation and condensation of uranium compounds in order to obtain an improved crystalline structure of this material. The apparatus comprises a vaporizing chamber and condensing structure connected thereto. There condenser is fitted with a removable liner having a demountable baffle attached to the liner by means of brackets and a removable pin. The baffle is of spiral cross-section and is provided with cooling coils disposed between the surfaces of the baffle for circulation of a temperature controlling liquid within the baffle. The cooling coll provides for controlllng the temperature of the baffle to insure formatlon of a satisfactory condensate, and the removable liner facilitates the removal of condensate formed during tbe sublimation process.

  13. The human chromosome. Electron microscopic observations on chromatin fiber organization.

    PubMed

    Abuelo, J G; Moore, D E

    1969-04-01

    Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 +/- 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25-50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.

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

    PubMed

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

    2006-06-01

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

  15. Cohesin's role as an active chromatin domain anchorage revealed.

    PubMed

    Feig, Christine; Odom, Duncan T

    2013-12-11

    Cohesin is a conserved protein complex indispensible for proper cell division, because it secures sister-chromatid cohesion following DNA replication until segregation is required at the onset of anaphase. Recent studies have revealed functions beyond this, showing that cohesin binds to interphase chromatin regulating gene expression at select loci via long-range chromosomal interactions. In this issue of The EMBO Journal, Sofueva et al (2013) use a combination of chromatin conformation capture methods, classical FISH imaging, and loss-of-function studies to elegantly demonstrate how cohesin controls the 3D architectural organization of the genome.

  16. Chromatin modification by the RNA Polymerase II elongation complex

    PubMed Central

    Tanny, Jason C.

    2014-01-01

    Transcription elongation by RNA polymerase II (RNAP II) involves the coordinated action of numerous regulatory factors. Among these are chromatin-modifying enzymes, which generate a stereotypic and conserved pattern of histone modifications along transcribed genes. This pattern implies a precise coordination between regulators of histone modification and the RNAP II elongation complex. Here I review the pathways and molecular events that regulate co-transcriptional histone modifications. Insight into these events will illuminate the assembly of functional RNAP II elongation complexes and how the chromatin landscape influences their composition and function. PMID:25494544

  17. Retention of the Native Epigenome in Purified Mammalian Chromatin

    PubMed Central

    Ehrensberger, Andreas H.; Franchini, Don-Marc; East, Philip; George, Roger; Matthews, Nik; Maslen, Sarah L.; Svejstrup, Jesper Q.

    2015-01-01

    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. PMID:26248330

  18. Water condensation: a multiscale phenomenon.

    PubMed

    Jensen, Kasper Risgaard; Fojan, Peter; Jensen, Rasmus Lund; Gurevich, Leonid

    2014-02-01

    The condensation of water is a phenomenon occurring in multiple situations in everyday life, e.g., when fog is formed or when dew forms on the grass or on windows. This means that this phenomenon plays an important role within the different fields of science including meteorology, building physics, and chemistry. In this review we address condensation models and simulations with the main focus on heterogeneous condensation of water. The condensation process is, at first, described from a thermodynamic viewpoint where the nucleation step is described by the classical nucleation theory. Further, we address the shortcomings of the thermodynamic theory in describing the nucleation and emphasize the importance of nanoscale effects. This leads to the description of condensation from a molecular viewpoint. Also presented is how the nucleation can be simulated by use of molecular models, and how the condensation process is simulated on the macroscale using computational fluid dynamics. Finally, examples of hybrid models combining molecular and macroscale models for the simulation of condensation on a surface are presented.

  19. Condensation in Nanoporous Packed Beds.

    PubMed

    Ally, Javed; Molla, Shahnawaz; Mostowfi, Farshid

    2016-05-10

    In materials with tiny, nanometer-scale pores, liquid condensation is shifted from the bulk saturation pressure observed at larger scales. This effect is called capillary condensation and can block pores, which has major consequences in hydrocarbon production, as well as in fuel cells, catalysis, and powder adhesion. In this study, high pressure nanofluidic condensation studies are performed using propane and carbon dioxide in a colloidal crystal packed bed. Direct visualization allows the extent of condensation to be observed, as well as inference of the pore geometry from Bragg diffraction. We show experimentally that capillary condensation depends on pore geometry and wettability because these factors determine the shape of the menisci that coalesce when pore filling occurs, contrary to the typical assumption that all pore structures can be modeled as cylindrical and perfectly wetting. We also observe capillary condensation at higher pressures than has been done previously, which is important because many applications involving this phenomenon occur well above atmospheric pressure, and there is little, if any, experimental validation of capillary condensation at such pressures, particularly with direct visualization.

  20. Epimerization in peptide thioester condensation.

    PubMed

    Teruya, Kenta; Tanaka, Takeyuki; Kawakami, Toru; Akaji, Kenichi; Aimoto, Saburo

    2012-11-01

    Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C-terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a thioester-thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester-thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively.

  1. Characterization of spacecraft humidity condensate

    NASA Technical Reports Server (NTRS)

    Muckle, Susan; Schultz, John R.; Sauer, Richard L.

    1994-01-01

    When construction of Space Station Freedom reaches the Permanent Manned Capability (PMC) stage, the Water Recovery and Management Subsystem will be fully operational such that (distilled) urine, spent hygiene water, and humidity condensate will be reclaimed to provide water of potable quality. The reclamation technologies currently baselined to process these waste waters include adsorption, ion exchange, catalytic oxidation, and disinfection. To ensure that the baseline technologies will be able to effectively remove those compounds presenting a health risk to the crew, the National Research Council has recommended that additional information be gathered on specific contaminants in waste waters representative of those to be encountered on the Space Station. With the application of new analytical methods and the analysis of waste water samples more representative of the Space Station environment, advances in the identification of the specific contaminants continue to be made. Efforts by the Water and Food Analytical Laboratory at JSC were successful in enlarging the database of contaminants in humidity condensate. These efforts have not only included the chemical characterization of condensate generated during ground-based studies, but most significantly the characterization of cabin and Spacelab condensate generated during Shuttle missions. The analytical results presented in this paper will be used to show how the composition of condensate varies amongst enclosed environments and thus the importance of collecting condensate from an environment close to that of the proposed Space Station. Although advances were made in the characterization of space condensate, complete characterization, particularly of the organics, requires further development of analytical methods.

  2. The evolutionary design of condensers

    NASA Astrophysics Data System (ADS)

    Bejan, A.; Lee, J.; Lorente, S.; Kim, Y.

    2015-03-01

    Condensers are flow architectures needed to provide high rates of condensation (or cooling) per unit volume, in enclosures with fixed volume. Their design has not changed from configurations consisting of the banks of horizontal tubes. In this paper, we outline a free path to evolving the design by exploring new features of flow configuration: flattened tubes, multiple tube sizes, arrays of flattened tubes, vertical tubes with turbulent film flow, forced convection condensation instead of gravity driven condensation, and the optimal length of a horizontal tube, i.e., the number of tubes in a column aligned with vapor cross flow. We show that the condensation density can be increased sizably by varying freely and without bias the morphology of the flow system: the shapes and arrangement of the cooled surfaces on which condensation occurs. The evolution of technology is described in terms of the special time direction of the useful (purposeful) changes in the configuration (shapes, arrangements) of surfaces on which flow/condensation occurs. This explains what "evolution" means. It is an important step for physics, not just technology.

  3. Condensation in Nanoporous Packed Beds.

    PubMed

    Ally, Javed; Molla, Shahnawaz; Mostowfi, Farshid

    2016-05-10

    In materials with tiny, nanometer-scale pores, liquid condensation is shifted from the bulk saturation pressure observed at larger scales. This effect is called capillary condensation and can block pores, which has major consequences in hydrocarbon production, as well as in fuel cells, catalysis, and powder adhesion. In this study, high pressure nanofluidic condensation studies are performed using propane and carbon dioxide in a colloidal crystal packed bed. Direct visualization allows the extent of condensation to be observed, as well as inference of the pore geometry from Bragg diffraction. We show experimentally that capillary condensation depends on pore geometry and wettability because these factors determine the shape of the menisci that coalesce when pore filling occurs, contrary to the typical assumption that all pore structures can be modeled as cylindrical and perfectly wetting. We also observe capillary condensation at higher pressures than has been done previously, which is important because many applications involving this phenomenon occur well above atmospheric pressure, and there is little, if any, experimental validation of capillary condensation at such pressures, particularly with direct visualization. PMID:27115446

  4. Steam generators, turbines, and condensers. Volume six

    SciTech Connect

    Not Available

    1986-01-01

    Volume six covers steam generators (How steam is generated, steam generation in a PWR, vertical U-tube steam generators, once-through steam generators, how much steam do steam generators make.), turbines (basic turbine principles, impulse turbines, reaction turbines, turbine stages, turbine arrangements, turbine steam flow, steam admission to turbines, turbine seals and supports, turbine oil system, generators), and condensers (need for condensers, basic condenser principles, condenser arrangements, heat transfer in condensers, air removal from condensers, circulating water system, heat loss to the circulating water system, factors affecting condenser performance, condenser auxiliaries).

  5. The chromatin remodelers RSC and ISW1 display functional and chromatin-based promoter antagonism.

    PubMed

    Parnell, Timothy J; Schlichter, Alisha; Wilson, Boris G; Cairns, Bradley R

    2015-01-01

    ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex or mutations in the 'basic patch' of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures.

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

    PubMed Central

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

    2016-01-01

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

  7. Effect of saffron on rat sperm chromatin integrity

    PubMed Central

    Mardani, Mohammad; Vaez, Ahmad; Razavi, Shahnaz

    2014-01-01

    Background: Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. Objective: The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Materials and Methods: Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining. Results: The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (p<0.001). Moreover, the AO staining results showed that the mean percentage of sperm with DNA damage was significantly decreased in saffron and vitamin E groups as compared with control group (p<0.001). Conclusion: Our results purposed that saffron can protect sperm against DNA damage and chromatin anomalies. PMID:25031579

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

    PubMed Central

    Exner, Vivien; Gruissem, Wilhelm; Hennig, Lars

    2008-01-01

    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. PMID:18477400

  9. Chromatin-regulating proteins as targets for cancer therapy

    PubMed Central

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, 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. PMID:24522270

  10. Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

    PubMed

    Adelman, Karen; Wei, Wenxiang; Ardehali, M Behfar; Werner, Janis; Zhu, Bing; Reinberg, Danny; Lis, John T

    2006-01-01

    The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. PMID:16354696

  11. Relative contributions of chromatin and kinetochores to mitotic spindle assembly

    PubMed Central

    Lončarek, Jadranka; Kaláb, Petr; Khodjakov, Alexey

    2009-01-01

    During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly. PMID:19805628

  12. Distinct Chromatin Modulators Regulate the Formation of Accessible and Repressive Chromatin at the Fission Yeast Recombination Hotspot ade6-M26

    PubMed Central

    Mizuno, Ken-ichi; Shibata, Takehiko; Ohta, Kunihiro

    2008-01-01

    Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) regulate transcription and recombination via alteration of local chromatin configuration. The ade6-M26 allele of Schizosaccharomyces pombe creates a meiotic recombination hotspot that requires a cAMP-responsive element (CRE)-like sequence M26, the Atf1/Pcr1 heterodimeric ATF/CREB transcription factor, the Gcn5 HAT, and the Snf22 SWI2/SNF2 family ADCR. Chromatin alteration occurs meiotically around M26, leading to the activation of meiotic recombination. We newly report the roles of other chromatin remodeling factors that function positively and negatively in chromatin alteration at M26: two CHD-1 family ADCRs (Hrp1 and Hrp3), a Spt-Ada-Gcn5 acetyltransferase component (Ada2), and a member of Moz-Ybf2/Sas3-Sas2-Tip60 family (Mst2). Ada2, Mst2, and Hrp3 are required for the full activation of chromatin changes around M26 and meiotic recombination. Acetylation of histone H3 around M26 is remarkably reduced in gcn5Δ, ada2Δ and snf22Δ, suggesting cooperative functions of these HAT complexes and Snf22. Conversely, Hrp1, another CHD-1 family ADCR, maintains repressive chromatin configuration at ade6-M26. Interestingly, transcriptional initiation site is shifted to a site around M26 from the original initiation sites, in couple with the histone acetylation and meiotic chromatin alteration induced around 3′ region of M26, suggesting a collaboration between these chromatin modulators and the transcriptional machinery to form accessible chromatin. These HATs and ADCRs are also required for the regulation of transcription and chromatin structure around M26 in response to osmotic stress. Thus, we propose that multiple chromatin modulators regulate chromatin structure reversibly and participate in the regulation of both meiotic recombination and stress-induced transcription around CRE-like sequences. PMID:18199689

  13. Coulomb interactions and fermion condensation

    SciTech Connect

    Capstick, S.; Cutkosky, R.E.; Joensen, M.A. ); Wang, K.C. )

    1990-08-15

    The influence of the Coulomb interaction in states containing massless and flavorless fermion-antifermion pairs is studied, using a continuum formulation within the finite volume {ital S}{sup 3}. Several different forms for the Coulomb interaction are examined, including confining potentials as well as nonconfining potentials. The calculations show that if the interaction is strong enough, the Coulomb interaction leads to condensation of pairs, and that this condensation has a chiral character. The condensation does not depend on whether the interaction is confining. It is found that simplified variational approximations are not accurate enough for an adequate description of the states.

  14. Neonatal hygroscopic condenser humidifier.

    PubMed

    Gedeon, A; Mebius, C; Palmer, K

    1987-01-01

    A hygroscopic condenser humidifier was developed for neonates on mechanical ventilation and was evaluated by laboratory tests and clinically. Humidification provided by the unit was measured in the 10- to 50-ml tidal-volume range at ambient temperatures of 24 degrees C and 38 degrees C. The effect of a leaking patient connection on device performance was investigated. Leakage rates were measured routinely in a neonatal ICU and surgery to determine the clinical significance. In the entire tidal volume and temperature range, the unit provided an inspiratory water content in excess of 30 g/m3 when the leak fraction (volume leaked/volume delivered at Y-piece) was less than 15%. This was found in three out of four cases. In about one out of ten cases, the leak exceeded 30%, which invariably led to corrective action, such as repositioning or changing the endotracheal tube. However, even at a 30% leak, a water content of about 26 g/m3 was still available for humidifying the inspired gas, which corresponds to normal physiologic conditions found in the trachea for nasal breathing of room air.

  15. Human Genome Replication Proceeds through Four Chromatin States

    PubMed Central

    Julienne, Hanna; Zoufir, Azedine; Audit, Benjamin; Arneodo, Alain

    2013-01-01

    Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a

  16. A thermal denaturation study of macronuclear chromatin in Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida).

    PubMed

    Salvini, M; Dalle Lucche, T; Durante, M

    1997-08-15

    The macronuclear chromatin of the ciliate Blepharisma japonicum, in two starvation states, was studied by thermal denaturation analysis. The behaviour of B. japonicum chromatin, native and reconstituted with Tetrahymena pyriformis H1 histone, was analysed. The data obtained are consistent with the hypothesis that B. japonicum macronuclear chromatin contains a H1-like peptide associated with the linker DNA, although this peptide is reduced in amount and/or chromatin stabilising ability when compared to Tetrahymena macronuclear H1.

  17. Using condenser performance measurements to optimize condenser cleaning

    SciTech Connect

    Wolff, P.J.; March, A.; Pearson, H.S.

    1996-05-01

    Because plant personnel perform condenser monitoring primarily to determine cleaning schedules, the accuracy and repeatability of a technique should be viewed within the context of a condenser cleaning schedule. Lower accuracy is acceptable if the cleaning schedule arising from that system is identical to a cleaning schedule arising from a technique with higher accuracy. Three condenser performance monitors were implemented and compared within the context of a condenser cleaning schedule to determine the relative advantages of different condenser monitoring techniques. These systems include a novel on-line system that consists of an electromagnetic flowmeter and an RTD mounted in a compact waterproof cylinder, an overall on-line system, and routine plant tests. The fouling measurements from each system are used in an optimization program which automatically computes a cleaning schedule that minitrack the combined cost of cleaning and the cost of increased fuel consumption caused by condenser fouling. The cleaning schedules resulting from each system`s measurements are compared. The optimization routine is also used to evaluate the sensitivity of optimal cleaning schedules to fouling rate and of the cost in dollars for non-optimal cleaning.

  18. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    PubMed

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

  19. Chromatin-modifying enzymes as modulators of reprogramming.

    PubMed

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

    2012-03-29

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

  20. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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